Xenobiotic Metabolism: In Vitro Methods 9780841204867, 9780841206274, 0-8412-0486-1

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Xenobiotic Metabolism: In Vitro Methods Gaylord D . Paulson, D . Stuart Frear, and E d w i n P. Marks, EDITORS U. S. Department of Agriculture

A symposium sponsored by the A C S Division of Pesticide Chemistry at the 176th Meeting of the American Chemical Society, Miami, Florida, September 10-15, 1978.

ACS SYMPOSIUM SERIES AMERICAN CHEMICAL SOCIETY 1979 WASHINGTON, D. C. In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Library of Congress CIP Data

Xenobiotic metabolism, in vitro methods. (ACS symposium series; 97 ISSN 0097-6156) Includes bibliographies and index. 1. Xenobiotic metabolism—Congresses. I. Paulson, Gaylord D. II. Frear, D. S., 1929III. Marks, Edwin P., 1925- . IV. American Chemical Society. Division of Pesticide Chemistry. V. Series: American Chemical Society. ACS symposium series; 97. QH521.X46 615.9'02 79-789 ISBN 0-8412-0486-1 ASCMC 8 97 1-328 1979

Copyright ©

1979

American Chemical Society A l l Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. P R I N T E D I N THE U N I T E D

STATES

AMERICA

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

ACS Symposium Series Robert F . G o u l d , Editor

Advisory Board Kenneth B. Bischoff

James P. Lodge

Donald G. Crosby

John L. Margrave

Robert E. Feeney

Leon Petrakis

Jeremiah P. Freeman

F. Sherwood Rowland

E. Desmond Goddard

Alan C. Sartorelli

Jack Halpern

Raymond B. Seymour

Robert A. Hofstader

Aaron Wold

James D. Idol, Jr.

Gunter Zweig

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

FOREWORD The A C S S Y M P O S I U M S E R I E S was founded in 1 9 7 4 to provide a medium for publishin format of the Serie I N C H E M I S T R Y SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PREFACE he beneficial effects of a wide variety of pesticides and other xenobiotics i n eliminating or controlling certain insects, plants, and disease processes have been demonstrated conclusively. The standard of living that we now enjoy is attributable, at least in part, to an increased use of xenobiotics. However, there is also a growing awareness and concern that some xenobiotics may have adverse effects on both man and his environment. This concern has resulted i n more extensive testing and evaluation of xenobiotics One type of information an understanding of the metabolic fate of the xenobiotic i n both target and nontarget organisms. In the past, most xenobiotic metabolism studies were conducted with the intact plant or animal. Such i n vivo studies have generated a wealth of useful information and w i l l continue to be the method of choice for many investigations. However, there is a growing realization that i n vitro studies may be superior for generating certain types of information. F o r example, in vitro techniques are often the methods of choice when there is a need to isolate and identify intermediate products of a multistep metabolic sequence. Cofactor requirements and other factors (inhibitors, activators, etc.) affecting the enzymes involved i n xenobiotic biotransformations usually are determined by i n vitro studies. Comparative studies to determine the effect of factors such as species, sex, age, tissue, subcellular fraction, nutritional factors, disease states, etc. on xenobiotic metabolism often are conducted most quickly and easily i n vitro. Usually the mode of action and selectivity of xenobiotics are investigated most effectively by i n vitro studies. Although i n vitro techniques are extremely useful and have broad application in studying the metabolic fate of xenobiotics, they also have definite limitations. F o r example, many i n vitro techniques are not applicable to long-term studies because of the buildup of end products, microbial contamination, and other problems. Some investigators have used i n vitro conditions (temperature, substrate concentrations, etc.) that had little or no relationship to conditions i n the intact organism; therefore, the results obtained were of limited value. Endogenous inhibitors that are retained in subcellular compartments (and therefore have no effect on the metabolism of a xenobiotic i n vivo) may be released K

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during the preparation of an i n vitro system and result i n misleading information. These and other potential problems cited i n the proceedings of this symposium make it clear that i n vitro methods must be used with caution. However, when properly used within the recognized limitations, in vitro techniques are extremely useful i n studying the metabolism of xenobiotics. This symposium was organized because of the growing interest i n the use of i n vitro techniques for xenobiotic metabolism studies. The primary objectives were to critically review, evaluate, and summarize: ( 1 ) how i n vitro techniques are used i n the laboratory with special emphasis on their application to xenobiotic metabolism studies; (2) advantages, disadvantages, and limitations of these techniques; and (3) examples of how i n vitro techniques may be useful i n future studies. It is our hope that the proceeding departure for a more effectiv future research on the metabolism and fate of xenobiotics i n the environment. Metabolism and Radiation Research Laboratory Agriculture Research,

G A Y L O R D D. P A U L S O N D. S T U A R T F R E A R

Science and Education Administration

E D W I N P. M A R K S

U.S. Department of Agriculture Fargo, ND 58102 December 20, 1978

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In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1 Xenobiotic Metabolism in Plants: In Vitro Tissue, Organ, and Isolated Cell Techniques R. H . S H I M A B U K U R O and

W.

C.

1

WALSH

Metabolism and Radiation Research Laboratory, Agricultural Research, Science and Education Administration, U . S. Department of Agriculture, Fargo, ND 58105

Interest i n xenobioti p r i m a r i l y on the f a t e o h e r b i c i d e s have been of predominant i n t e r e s t i n p l a n t metabol i s m s t u d i e s , the methods and techniques discussed i n t h i s report are e q u a l l y a p p l i c a b l e to other c l a s s e s of p e s t i c i d e s i n c l u d i n g i n s e c t i c i d e s and f u n g i c i d e s . In t h i s r e p o r t , the term " x e n o b i o t i c s " r e f e r s to s y n t h e t i c p e s t i c i d e s and not to other unnatural compounds. However, the d i s c u s s i o n on i n v i t r o techniques f o r x e n o b i o t i c metabolism i n p l a n t s i s based p r i m a r i l y on research with h e r b i c i d e s . The metabolism of p e s t i c i d e s i n p l a n t s i s discussed e x t e n s i v e l y i n s e v e r a l p u b l i c a t i o n s (1, _2, 3). Much i s known on the metabolism of organic p e s t i c i d e chemicals i n p l a n t s , but the f a t e of most p e s t i c i d e s i n p l a n t s i s s t i l l unknown. I t i s important to know the i d e n t i t y of t r a n s i t o r y intermediate products and the u l t i m a t e f a t e of these chemicals i n p l a n t s s i n c e the intermediate products may be t o x i c . Knowledge of chemical i d e n t i t y and quantity of intermediate products of a h e r b i c i d e i n p l a n t s at d i f f e r e n t times a f t e r treatment i s e s s e n t i a l f o r the e l u c i d a t i o n of the mode of a c t i o n and b a s i s for s e l e c t i v i t y . Whole plants t r e a t e d with p e s t i c i d e s through t h e i r roots or f o l i a g e have been used e x t e n s i v e l y f o r metabolism and " t e r m i n a l " residue s t u d i e s . U s e f u l q u a n t i t i e s of metabolites may be generated from l a r g e - s c a l e treatments of whole p l a n t s ; such metabolites can then be p u r i f i e d f o r chemical c h a r a c t e r i z a t i o n . The development of v a r i o u s chromatographic techniques and improved UV, IR, NMR and mass spectroscopy instrumentation 1

Mention of a trademark or p r o p r i e t a r y product does not c o n s t i t u t e a guarantee or warranty of the product by the U. S. Department of A g r i c u l t u r e and does not imply i t s approval to the e x c l u s i o n of other products that may a l s o be s u i t a b l e . This chapter not subject to U.S. copyright. Published 1979 American C h e m i c a l Society

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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has made i t p o s s i b l e to c h a r a c t e r i z e chemically small amounts of metabolites. Excised p l a n t t i s s u e s and organs and i s o l a t e d c e l l s may be used f o r p e s t i c i d e metabolism s t u d i e s f o r short periods where l i m i t e d q u a n t i t i e s o f metabolites a r e generated. Results from s e l e c t e d r e p o r t s are presented t o i l l u s t r a t e the techniques and methods that may be used. Metabolism of X e n o b i o t i c s The s u c c e s s f u l i s o l a t i o n and chemical c h a r a c t e r i z a t i o n of any x e n o b i o t i c b i o t r a n s f o r m a t i o n product r e q u i r e the generation of s u f f i c i e n t q u a n t i t i e s o f the metabolite. The degradation mechanisms i n p l a n t s may be slower than those i n animals (_1). P l a n t s a l s o l a c k an excretory system comparable to the r e n a l e x c r e t i o n system i n mammals t i o n products o f p e s t i c i d e e x c r e t i o n products as with mammals. P l a n t s metabolize s i g n i f i cant amounts o f p e s t i c i d e s u l t i m a t e l y t o i n s o l u b l e residues ( 3 ) . The chemical nature and q u a n t i t i e s of the metabolites i n a p l a n t are i n f l u e n c e d by the s i t e of absorption of the p e s t i c i d e , t r a n s l o c a t i o n , and the residence time i n the p l a n t . The use of excised plant t i s s u e s , organs, and i s o l a t e d c e l l s f o r s t u d i e s of x e n o b i o t i c metabolism i s an attempt to modify the i n f l u e n c e of the above p h y s i o l o g i c a l f u n c t i o n s i n order to optimize the c o n d i t i o n s f o r maximum metabolite generation. Fundamental f u n c t i o n s of the whole p l a n t , i n c l u d i n g absorption, t r a n s l o c a t i o n , c e l l f u n c t i o n s and senescence should be considered when i n v i t r o techniques with i s o l a t e d p l a n t parts are used. The p h y s i o l o g i c a l s i g n i f i c a n c e of metabolism i n i s o l a t e d plant p a r t s must be evaluated u l t i m a t e l y i n terms of r e s u l t s i n intact plants. Absorption. Regardless o f how a p e s t i c i d e i s a p p l i e d to the p l a n t , the chemical must penetrate the p l a n t and be absorbed s p e c i f i c a l l y i n t o the c e l l s where b i o t r a n s f o r m a t i o n r e a c t i o n s occur. The l e a f surfaces and root t i p s are the primary s i t e s o f p e n e t r a t i o n i n t o the plant (4, 5 ) . The c u t i c l e , a t h i n , l i p o i d a l membrane that covers the e n t i r e s u r f a c e area of the above ground p a r t s o f a p l a n t , i s the primary b a r r i e r to p e n e t r a t i o n by organic p e s t i c i d e s . The p e n e t r a t i o n o f nonpolar organic p e s t i c i d e s i n t o leaves and roots i s b e l i e v e d to be a two-stage process (4, 6 ) . The f i r s t stage i n l e a f a b s o r p t i o n i n v o l v e s passive p e n e t r a t i o n or p a r t i t i o n i n g o f the nonpolar compound i n t o the c u t i c l e and d e s o r p t i o n i n t o the c e l l w a l l s (apoplast) o f the u n d e r l y i n g cells. In r o o t s , the f i r s t stage i n v o l v e s the i n a c t i v e d i f f u s i o n of the compound i n t o the root " f r e e space" ( a p o p l a s t ) . The second stage i n leaves i s the a c t i v e transport of the p e s t i c i d e s across the plasmalemma ( c e l l membrane) i n t o l e a f c e l l s (symplast), and i n some cases i n t o the phloem f o r

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Plant Tissues, Organs, and Isolated Cells

symplastic t r a n s p o r t . Symplastic transport i s an a c t i v e , energy-requiring process o c c u r r i n g i n the l i v i n g cytoplasmic continuum of the p l a n t . In r o o t s , the second stage i n v o l v e s an a c t i v e transport o f the p e s t i c i d e s across the endodermis and i n t o the s t e l e where the d i f f e r e n t i a t e d v a s c u l a r s t r u c t u r e i s l o c a t e d . Therefore, symplastic i n t e r c e l l u l a r t r a n s p o r t of p e s t i c i d e s occurs a t one stage during the t r a n s p o r t of p e s t i c i d e s from the e x t e r n a l root s o l u t i o n i n t o the xylem v e s s e l s i n the s t e l e f o r a p o p l a s t i c transport to the shoot. Apoplastic transport from the root t o shoot i s an i n a c t i v e , p h y s i c a l process o c c u r r i n g i n the n o n - l i v i n g extra-protoplasmic component o f the p l a n t under the i n f l u e n c e o f the t r a n s p i r a t i o n stream. The symplast-apoplast concept i s a p p l i e d as defined by C r a f t s and C r i s p (7) Unfortunately l i t t l e i s known about the mechanisms o f absorptio molecules i n r o o t s . Th information regarding the absorption o f i n o r g a n i c ions by r o o t s . T r a n s l o c a t i o n . Once p e n e t r a t i o n i n t o l e a f c e l l s or root t i p s i s accomplished, the p e s t i c i d e must be t r a n s l o c a t e d s y m p l a s t i c a l l y from leaves and a p o p l a s t i c a l l y from roots t o d i f f e r e n t plant organs and d i s t r i b u t e d to s p e c i f i c t i s s u e s and c e l l s where the t a r g e t s i t e s f o r b i o l o g i c a l a c t i v i t y may be l o c a t e d . The t a r g e t s i t e s o f the p e s t i c i d e may o r may not be l o c a t e d i n the same t i s s u e s or c e l l s as the b i o t r a n s f o r m a t i o n s i t e s . D e t a i l e d d i s c u s s i o n s on s t r u c t u r e of the v a s c u l a r system and mechanisms of t r a n s p o r t are discussed i n s e v e r a l p u b l i c a t i o n s (5, 8). I t i s g e n e r a l l y recognized that phloem or symplastic t r a n s p o r t occurs from "sources to s i n k s " or i n broad terms from green leaves t o a c t i v e centers of growth and storage. This r e s u l t s i n d i f f e r e n t i a l or s e l e c t i v e t r a n s l o c a t i o n and accumulation of photosynthates i n young leaves, buds, and meristematic regions of the p l a n t . Most h e r b i c i d e s , and probably other p e s t i c i d e s , do not appear to t r a n s l o c a t e very r e a d i l y i n the symplast (2) although exceptions a r e known ( 5 ) . N o n s p e c i f i c t r a n s l o c a t i o n and uniform d i s t r i b u t i o n to a l l p a r t s of p l a n t s occur by a p o p l a s t i c t r a n s p o r t . This i s g e n e r a l l y observed when h e r b i c i d e s are t r a n s l o c a t e d i n the xylem from roots to shoots under the i n f l u e n c e o f the t r a n s p i r a t i o n stream (9). The rates of p e s t i c i d e s t r a n s l o c a t e d i n the xylem appear to depend on the amount of m a t e r i a l r e l e a s e d by parenchyma c e l l s to the xylem (9). Factors a f f e c t i n g t r a n s p i r a t i o n a l s o i n f l u e n c e a p o p l a s t i c t r a n s p o r t of p e s t i c i d e s from roots t o shoots. The a b s o r p t i o n and t r a n s l o c a t i o n functions i n a whole p l a n t r e f l e c t the f u n c t i o n s o f s p e c i f i c organs, t i s s u e s , and c e l l s organized and i n t e g r a t e d i n t h e i r a c t i v i t i e s t o meet the requirements of growth and maintenance. The i m p l i c a t i o n s o f s e p a r a t i n g the organs, t i s s u e s , and c e l l s f o r use i n i n v i t r o

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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x e n o b i o t i c metabolism s t u d i e s are c l e a r l y

M E T A B O L I S M

evident.

Senescence. Senescence of p l a n t t i s s u e s i s a major f a c t o r that must be considered i n x e n o b i o t i c metabolism s t u d i e s w i t h i s o l a t e d p l a n t organs and c e l l s . In c o n t r a s t to mammals, senescence i n p l a n t s i s not due to i r r e v e r s i b l e changes i n the genome (DNA breakdown), but due to i n t e r n a l p l a n t f a c t o r s that i n h i b i t c e l l metabolism or a l t e r i t s d i r e c t i o n toward a u t o l y t i c pathways (10). Plant senescence i s a hormonally c o n t r o l l e d phenomenon (10, 11) that may be induced, retarded or reversed under d i f f e r e n t circumstances. The process of l e a f senescence begins as soon as leaves are excised or detached from the whole p l a n t . P r o t e i n synthe­ s i s and c h l o r o p h y l l conten r e s p i r a t i o n , and RNase (12, 13, 14, 15). In whea lipase, esterase, and a c i d phosphatase d e c l i n e d a f t e r detachment. However, the d e c l i n e i n the enzyme l e v e l s was retarded by treatment of leaves w i t h k i n e t i n (15). L i g h t r e t a r d s senes­ cence i n excised leaves (12). Light-induced r e t a r d a t i o n of senescence was not l i n k e d to phytochrome a c t i o n , but was r e l a t e d d i r e c t l y to photosynthesis (12). However, evidence i n d i c a t e s that l i g h t r e t a r d a t i o n of senescence i s not l i n k e d to CO2 f i x a t i o n or photochemical a c t i v i t y of PS I I . Diuron [ 3 - ( 3 , 4 - d i c h l o r o p h e n y l ) - l , l - d i m e t h y l u r e a ] d i d not e l i m i n a t e or reduce the e f f e c t i v e n e s s of l i g h t i n r e t a r d i n g c h l o r o p h y l l l o s s (16). A d d i t i o n of sucrose a l s o had no e f f e c t on c h l o r o p h y l l l o s s when CO2 f i x a t i o n was i n h i b i t e d by diuron (16). Glucose i n h i b i t e d s i g n i f i c a n t l y the l o s s of c h l o r o p h y l l i n the dark (14). However, the e f f e c t i v e n e s s of glucose at the optimum concentration of 100 μΜ was s t i l l only h a l f the e f f e c t i v e n e s s of k i n e t i n at 10 μΜ (14). I f a high-energy intermediate i s r e q u i r e d to delay senescence, the r e s u l t s i n d i c a t e that l i g h t may be a c t i n g through i t s a c t i o n on c y c l i c photophosphorylat i o n , a system that i s not i n h i b i t e d by diuron. T o t a l p r o t e i n s y n t h e s i s d e c l i n e s i n senescence but a s p e c i f i c proteinase with L - s e r i n e i n i t s a c t i v e center i n ­ creases i n a c t i v i t y as senescence progresses (17). Most of the t o t a l s o l u b l e p r o t e i n l o s t i n e a r l y senescence was account­ ed f o r by a decrease i n r i b u l o s e - l , 5 - b i p h o s p h a t e carboxylase (13). Therefore, the c h l o r o p l a s t appears to be the o r g a n e l l e i n which the i n i t i a l senescence sequence begins. Excised leaves or l e a f d i s c s have been u t i l i z e d exten­ s i v e l y to study senescence i n p l a n t s . These systems have two advantages: 1) detached t i s s u e s senesce at a f a s t e r r a t e than when they are attached to the p l a n t , and 2) r e g u l a t o r y compounds can be fed conveniently to the t i s s u e s through the cut s u r f a c e s . However, i t i s u n c e r t a i n whether the biochemical and p h y s i o l o g ­ i c a l changes i n an excised l e a f resemble those i n attached leaves. These c o n s i d e r a t i o n s i n the study of senescence are

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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SHiMABUKURo

A N D W A L S H

Plant Tissues, Organs, and Isolated Cells

r e l e v a n t a l s o to s t u d i e s on metabolism o f x e n o b i o t i c s i n i s o l a t e d plant t i s s u e s . I s o l a t e d Plant Part and C e l l Methods Whole p l a n t s with p e s t i c i d e s a p p l i e d through t h e i r roots or leaves or i n j e c t e d i n t o stems and f r u i t s are the most commonly used experimental m a t e r i a l f o r x e n o b i o t i c metabolism s t u d i e s . However, s t u d i e s with i n t a c t p l a n t s are complicated by v a r i a b l e s r e l a t e d to root and l e a f absorption, t r a n s l o c a t i o n and t r a n s p i r a t i o n . To overcome some of these v a r i a b l e s , researchers have used excised p l a n t p a r t s and enzymatically separated mesophyll c e l l s . Isolated plant protoplasts also may be u s e f u l f o r x e n o b i o t i metabolis s t u d i e s Results of x e n o b i o t i biochemical and p h y s i o l o g i c a response separate plan parts or c e l l s a r e u s u a l l y extrapolated to r e f l e c t r e a c t i o n s o c c u r r i n g i n complex whole p l a n t s . T h i s may o r may not be appropriate and c a u t i o n must be e x e r c i s e d i n e v a l u a t i n g r e s u l t s from separated systems. The d i f f e r e n t methods used f o r xenob i o t i c metabolism s t u d i e s with plant p a r t s and i s o l a t e d c e l l s together with r e s u l t s from s e l e c t e d r e p o r t s are presented here. Excised Leaves and Roots. Whole organs separated from the i n t a c t p l a n t are used i n t h i s method. The leaves of both d i - and monocotyledonous p l a n t s may be used. Method. Dicotyledonous p l a n t s such as cotton (Gossypium hirsutum L.) (18, 19, 20), peanut (Arachis hypogaea L.) (21, 22), c a r r o t (Daucus c a r o t a L.) (20) and soybean [ G l y c i n e max (L.) Merr.] are grown u n t i l t h e i r f i r s t true leaves a r e f u l l y expanded. The p e t i o l e s of the true leaves are excised under water to prevent d i s r u p t i o n o f the water column i n the xylem by the i n t r o d u c t i o n of a i r . This precautionary step minimizes permanent w i l t i n g o f leaves due to the i n t e r r u p t i o n of the t r a n s p i r a t i o n stream. Leaves of monocotyledonous p l a n t s l a c k p e t i o l e s . Theref o r e , s e l e c t e d leaves may be excised near the base of the lamina or blade as described f o r the p e t i o l e s of d i c o t s . Leaves from corn (Zea mays L . ) , sorghum (Sorghum vulgare P e r s . ) , and sugarcane (Saccharum o f f i c i a n a r u m L.) have been excised and used s u c c e s s f u l l y (23, 24). Excised l e a f blades o f b a r l e y (Hordeum vulgare L.) and r i c e (Oryza s a t i v a L.) have been used to study the metabolism of detergents (25). The e n t i r e shoot of young (2- to 3-leaf stage) b a r l e y , wheat ( T r i t i c u m aestivum L.) and w i l d oat (Avena f a t u a L.) seedlings may be excised a t the s o i l l e v e l by the same method and used f o r metabolism s t u d i e s . M o r p h o l o g i c a l l y , the shoots o f young c e r e a l s and other grasses c o n s i s t o f overlapping l e a f sheaths o f emerged and younger leaves. The v a s c u l a r anatomy of the l e a f sheath

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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M E T A B O L I S M

would be comparable to that of the p e t i o l e . E x c i s i n g the shoot of young c e r e a l p l a n t s i s n e a r l y equivalent to e x c i s i n g s e v e r a l l e a f blades. The cut edges of e i t h e r p e t i o l e s or l e a f blades are immersed i n a s o l u t i o n of the x e n o b i o t i c compound (18, 25). Excised leaves are normally t r e a t e d i n a controlled-environment chamber with a d e f i n i t e photoperiod. L i g h t not only r e t a r d s senescence but i t s t i m u l a t e s t r a n s p i r a t i o n and increases the uptake of treatment s o l u t i o n . Excised whole r o o t s , separated from the shoots, have not been used e x t e n s i v e l y f o r metabolism s t u d i e s . The r o o t , a h e t e r o t r o p h i c p l a n t organ, i s more conducive f o r use i n t i s s u e c u l t u r e where a carbon source may be provided. Corn roots s u p p l i e d with glucose i n a s e p t i c c u l t u r e metabolized a t r a z i n e (2-chloro~4-ethylamino-6-isopropylamino-^-triazine hr p e r i o d (26). Excise corn, wheat, soybean, oats (Avena s a t i v a L.) and b a r l e y were incubated with simazine [2-chloro-4,6-bis (ethylamino)-s_t r i a z i n e ] i n Hoagland s n u t r i e n t s o l u t i o n f o r 6 hours (27). Excised roots and hypocotyls of soybean were incubated f o r 24 hours i n the dark i n a 0.1% Na2C03 or d i s t i l l e d water s o l u t i o n of amiben (3-amino-2,5-dichlorobenzoic a c i d ) . The amiben metabolite, N-glucosyl amiben [N-(3-carboxy-2,5-dichlorophenyl)glucosylamine], was i s o l a t e d and c h a r a c t e r i z e d from these t i s s u e s (28). Roots of dicotyledonous seedlings with l a r g e endogenous sources of carbon i n t h e i r cotyledons may be c u l t u r e d successf u l l y a f t e r removal of t h e i r e p i c o t y l s (immature shoots). The growth of roots from pea (Pisum sativum L.) seedlings with t h e i r e p i c o t y l s removed was s i m i l a r to growth of roots from i n t a c t seedlings f o r 11 days i n n u t r i e n t s o l u t i o n (29). These r o o t s were t r e a t e d with a t r a z i n e f o r 9 days. The endogenous reserve i n the cotyledons was the only carbon source f o r the roots. f

Discussion. The use of excised leaves f o r x e n o b i o t i c metabolism has s e v e r a l advantages: 1) ease of t r e a t i n g plant m a t e r i a l with the x e n o b i o t i c chemical, 2) r a p i d uptake of the chemical i n t o plant t i s s u e s , and 3) e l i m i n a t i o n of root and l e a f surface absorption as b a r r i e r s . Some l i m i t a t i o n s of t h i s technique i n c l u d e : 1) the treatment period f o r metabolism must be short, 2) senescence of the plant organ begins upon e x c i s i o n from the i n t a c t p l a n t , and 3) r e a c t i o n s i n excised plant organ may not be the same as those o c c u r r i n g i n i n t a c t p l a n t s . A r e l a t i v e l y l a r g e number of excised leaves can be t r e a t e d with a minimum volume of treatment s o l u t i o n to obtain maximum uptake and d i s t r i b u t i o n of a x e n o b i o t i c i n l e a f t i s s u e s . Metabolites of f l u o r o d i f e n ( 2 , 4 - d i n i t r o - 4 - t r i f l u o r o m e t h y l diphenylether) (22) and p e r f l u i d o n e [ l , 1 , 1 - t r i f l u o r o - N - { 2 methyl-4-(phenysulfonyl)phenyl}methanesulfonamide] (21) were 1

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

SHIMABUKURO

A N D W A L S H

Vlant Tissues, Organs, and

Isolated Cells

i s o l a t e d and c h a r a c t e r i z e d from 300 and 3000 excised peanut leaves, r e s p e c t i v e l y . Treatment s o l u t i o n s were prepared at p h y s i o l o g i c a l concentrations of 1 to 100 μΜ and s p a r i n g l y s o l u b l e compounds may be prepared as aqueous s o l u t i o n s c o n t a i n ­ ing up to 1% acetone (22). Acetone at 1% d i d not cause severe i n j u r y to excised leaves. The use of s u r f a c t a n t s and emulsif i e r s common to l e a f s u r f a c e a p p l i c a t i o n s i s not necessary. Absorption and transport of x e n o b i o t i c s to c e l l u l a r b i o ­ transformation s i t e s are f a i r l y r a p i d i n excised leaves. Absorption by d i c o t leaves i s r a p i d , u s u a l l y w i t h i n the f i r s t 3 to 5 hours of treatment. Peanut leaves absorbed approxi­ mately 3 to 4 ml of f l u o r o d i f e n s o l u t i o n per l e a f w i t h i n 5 hours (22), and cotton leaves absorbed up to 5 ml of c i s a n i l i d e ( c i s - 2 , 5 - d i m e t h y l - l - p y r r o l i d i n e c a r b o x a n i l i d e ) s o l u t i o n per l e a f w i t h i n 3 hours (20). A d d i t i o n a maintain excised leaves hours. The d i s t i l l e d water may be added as a pulse-chase and to replace treatment s o l u t i o n l o s t through t r a n s p i r a t i o n by excised leaves. Generally, the r a t e of x e n o b i o t i c absorption d e c l i n e s s i g n i f i c a n t l y a f t e r the i n i t i a l 3 to 5 hours. Absorption of treatment s o l u t i o n by excised l e a f blades of monocots i s not as r a p i d as with excised leaves of d i c o t s . The r a t e of treatment s o l u t i o n l o s t per u n i t l e a f area was not determined, but excised l e a f blades g e n e r a l l y r e q u i r e d l i t t l e a d d i t i o n a l d i s t i l l e d water. S u f f i c i e n t q u a n t i t i e s of jst r i a z i n e s (23), propachlor (2-chloro-N-isopropylacetamide) (24), and diclofop-methyl [methyl-2-{4-(2 ,4 -dichlorophenoxy) phenoxy}propanoate] (30) were absorbed w i t h i n 24- to 48-hour periods by excised corn and sorghum leaves and wheat shoots, r e s p e c t i v e l y , f o r metabolite i s o l a t i o n and c h a r a c t e r i z a t i o n . Excised leaves may be u s e f u l f o r i n v e s t i g a t i n g p e s t i c i d e i n t e r a c t i o n s . Rapid pulse-chase treatment of excised leaves i s p o s s i b l e with a x e n o b i o t i c preceded o r followed by a second compound. The pulse-chase technique with excised leaves a l s o i s u s e f u l i n s t u d i e s on product-precursor r e l a t i o n s h i p s . I s o l a t e d metabolites may be used i n treatments. Simultaneous treatment with x e n o b i o t i c s may be appropriate i f the compounds are compatible as a mixture. However, before any treatments with two or more compounds are made, the absorption and t r a n s l o ­ c a t i o n c h a r a c t e r i s t i c s of each compound i n excised leaves should be determined to i n s u r e proper e v a l u a t i o n of r e s u l t s . f

f

Absorption and t r a n s l o c a t i o n i n excised leaves. Absorp­ t i o n of a x e n o b i o t i c i n s o l u t i o n occurs predominantly through the cut edges of p e t i o l e s or l e a f blades or sheaths. There­ f o r e , any i n f l u e n c e exerted by roots on the absorption and t r a n s l o c a t i o n of a x e n o b i o t i c i s circumvented by the excised l e a f method. This method allows d i r e c t uptake of the x e n o b i o t i c i n t o the xylem (apoplast) and t r a n s p o r t throughout the l e a f under the i n f l u e n c e of the t r a n s p i r a t i o n stream. Maximum

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9

XENOBIOTIC

10

M E T A B O L I S M

number of degradation s i t e s i n the mesophyll c e l l s o f leaves should be exposed to the x e n o b i o t i c . This i s o f t e n a d i s t i n c t advantage of the excised l e a f method. However, the a p o p l a s t i c t r a n s p o r t of x e n o b i o t i c s , even when introduced d i r e c t l y i n t o the xylem, may not be s o l e l y a f u n c t i o n of t r a n s p i r a t i o n . Several h e r b i c i d e s were s e l e c t e d (Figure 1) to i l l u s t r a t e t r a n s l o c a t i o n d i f f e r e n c e s i n excised leaves. Figures 2 to 5 show the t r a n s l o c a t i o n of [-^C-ring] a m i t r o l e (3-amino-l,2,4-triazole) (A), [ ^ C - r i n g l a t r a z i n e (B), [ C - p h e n y l ] d i c l o f o p - m e t h y l (C), and [ C F ] f l u o r o d i f e n (D) i n r o o t - t r e a t e d i n t a c t p l a n t s and excised leaves o f soybean and oat. A l l h e r b i c i d e s were a p p l i e d a t 10 μΜ (spec. a c t . 0.54 mCi/mmol) concentration. Two excised soybean leaves and oat shoots were t r e a t e d with 10 l d 5 ml r e s p e c t i v e l y f h h e r b i c i d e f o r 24 hours soybean and oat p l a n t wer , r e s p e c t i v e l y , of each h e r b i c i d e f o r 48 hours (Figures 3 and 5). A l l p l a n t m a t e r i a l s were exposed to a 14-10-hour l i g h t - d a r k c y c l e a t 13 klux l i g h t i n t e n s i t y , 26 C day and 20 C night temperatures. The r e l a t i v e absorption date of soybean and oat p l a n t s (Tables I and I I ) cannot be compared d i r e c t l y s i n c e the surface areas and the l ^ C a p p l i e d d i f f e r e d between the two s p e c i e s . However, apparent d i f f e r e n c e s between a d i c o t (soy­ bean) and monocot (oat), as discussed p r e v i o u s l y , are evident. 14

l 5

3

Table I .

Absorption and t r a n s l o c a t i o n of [14c]herbicides b y excised soybean leaves and excised shoots of oat.u/

Herbicide Amitrole(A) Atrazine(B) Diclofopme thy1(C) Fluorodifen(D)

t

% of applied 14c absorbed^/

D i s t r i b u t i o n o f absorbed oat soybean

soybean

immerseaSJ

oat

1 4

l e a f immersedSJ

C(%)

shoot

99 99

32 28

6 1

94 99

53 22

47 78

92 94

77 56

36 49

64 51

75 81

25 19

2-free c o n d i t i o n s . A f l u x of a chemical i n t o and out of l e a f d i s c s may be expected (41, 51). The k i n e t i c s of uptake and e f f l u x of none l e c t r o l y t e s have been described (41). However, i n most of these studies i t was assumed that the chemical was not a l t e r e d between i t s uptake and e f f l u x by l e a f d i s c s . Some evidence i n d i c a t e s that t h i s may not be n e c e s s a r i l y t r u e . The e f f l u x from monuron-resistant p l a n t a i n l e a f d i s c s was n e a r l y one-half of the i n i t i a l l y absorbed h e r b i c i d e w i t h i n 2 to 3 hours (54). Subsequent reabsorption and metabolism occurred but 37% of the t o t a l 14c was present i n the e f f l u x a f t e r 7.6 hours. More than 50% of the l ^ c i n the e f f l u x was three metabolites with parent monuron accounting f o r the remainder (54). However, p o l a r water-soluble conjugates, which c o n s t i t u t e the major metabolites of monuron i n l e a f d i s c s , were not detected i n the e f f l u x . S i m i l a r l y , major water-soluble conjugates of a t r a z i n e (56) i n corn l e a f d i s c s and diclofop-methyl (30) i n wheat l e a f s e c t i o n s were not detected i n the e f f l u x from these t i s s u e s . Therefore, a s e l e c t i v e e f f l u x of non-polar parent compounds or t h e i r metabolites appears to occur i n l e a f d i s c s or s e c t i o n s with some x e n o b i o t i c s . Any r e l a t i o n s h i p between the e f f l u x c h a r a c t e r i s t i c s of l e a f d i s c s and r e l e a s e of m a t e r i a l from parenchyma c e l l s i n t o the v a s c u l a r system of i n t a c t p l a n t s i s s t i l l obscure. However, e f f l u x c h a r a c t e r i s t i c s of s p e c i f i c compounds from l e a f d i s c s may be r e l a t e d to t h e i r immobilization or t r a n s l o c a t i o n i n i n t a c t plants. The e v a l u a t i o n of x e n o b i o t i c metabolism as a f a c t o r i n s e l e c t i v i t y , antagonism, or synergism i s p o s s i b l e because of the r a p i d determination of metabolism i n l e a f d i s c s . Metabolism and

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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d e t o x i c a t i o n of h e r b i c i d e s i n p l a n t s i s probably the most important s i n g l e f a c t o r i n h e r b i c i d e s e l e c t i v i t y . P h y s i o l o g i c a l responses coupled to time-course metabolism may be r e a d i l y demonstrated i n l e a f d i s c s f o r photosynthetic i n h i b i t o r s such as monuron (55) and a t r a z i n e (44, 48, 56). I n h i b i t i o n of photosynthesis and recovery w i t h i n 8 hours was c o r r e l a t e d w i t h g l u t a t h i o n e conjugation of a t r a z i n e i n sorghum (44), corn (56), and s e v e r a l species of the s u b f a m i l i e s , Festucoideae and Panicoideae (48). Recovery of photosynthesis to n e a r l y the c o n t r o l l e v e l occurred i n monuron-treated cotton l e a f d i s c s w i t h i n 4 to 5 hours with r a p i d N-demethylation of monuron (55). The nature of i n t e r a c t i o n between x e n o b i o t i c s at the molecular l e v e l may be demonstrated r e a d i l y i n l e a f d i s c s . The a p p l i c a t i o n of a mixture of the h e r b i c i d e p r o p a n i l (3,4 dichloropropionanilide) c i d e caused i n j u r y to r e s i s t a n to the i n h i b i t i o n of an a r y l acylamidase i n r i c e by carbamate i n s e c t i c i d e s that prevented d e t o x i c a t i o n of p r o p a n i l i n the r e s i s t a n t species (58, 59). In r e s i s t a n t cotton l e a f d i s c s , c e r t a i n carbamate i n s e c t i c i d e s such as c a r b a r y l (1-napthylmethylcarbamate) s t r o n g l y i n h i b i t e d the degradation of the photosynthetic i n h i b i t o r , monuron. The i n h i b i t i o n of photo­ s y n t h e s i s was enhanced and normal recovery to c o n t r o l l e v e l s of photosynthetic a c t i v i t y was delayed (55). The i n t e r a c t i o n s between s e v e r a l organophosphate and carbamate i n s e c t i c i d e s and h e r b i c i d e s were t e s t e d i n l e a f d i s c s of s e v e r a l species (60, 61). Organophosphate i n s e c t i c i d e s , dyfonate ( 0-ethyl - S_- ph eny 1 ethylphosphonodithioate) and malat h i o n [£,0-dimethyl-S-(l,2-bis-carbethoxy)-ethyl phosphorod i t h i o a t e j , s t r o n g l y i n h i b i t e d the metabolism of s u b s t i t u t e d phenylurea h e r b i c i d e s . Several carbamates [ c a r b a r y l , carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate), ΡCMC (£-chlorophenyl-N-methylcarbamate)] s t r o n g l y i n h i b i t e d p r o p a n i l metabolism (60). The e f f e c t s of s e v e r a l h e r b i c i d e s on the degradation of c a r b a r y l , dyfonate, and malathion were not as severe as the e f f e c t s of the i n s e c t i c i d e s on h e r b i c i d e metabolism (61). Carbaryl metabolism was stimulated by c h l o r ­ propham while metabolism of dyfonate and malathion was i n h i b i t e d by p r o p a n i l (61). The nature of the i n t e r a c t i o n between carbofuran and the h e r b i c i d e s , alachor (2-ehloro-2 ,6'-diethyl-Nmethoxymethyl a c e t a n i l i d e ) (62), chlorbromuron [3-(4-bromo-3chlorophenyl)-l-methoxy-l-methylurea] (63), and b u t y l a t e (Se t h y l d i i s o b u t y l t h i o c a r b a m a t e ) (64) was explained on the b a s i s of whole-plant experiments. Carbofuran increased the absorption and i n h i b i t e d s l i g h t l y the metabolism of the h e r b i c i d e s . I f the i n h i b i t i o n of h e r b i c i d e metabolism by carbofuran i s s i g n i ­ f i c a n t , i t should be r e a d i l y demonstrated i n l e a f d i s c s over much shorter periods than i s p o s s i b l e with whole p l a n t s . 1

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

S H I M A B U K U R O A N D W A L S H

Phnt

Tissues, Organs, and Isolated Cells

23

I s o l a t i o n and c h a r a c t e r i z a t i o n o f metabolites. The i d e n t i f i c a t i o n of metabolites e x t r a c t e d from l e a f d i s c s i s g e n e r a l l y based on t h i n - l a y e r cochromatography with known standards. L i m i t e d numbers of l e a f d i s c s or s e c t i o n s are t r e a t e d with r a d i o l a b e l e d x e n o b i o t i c s , e x t r a c t e d , and the r a d i o ­ a c t i v e metabolites a r e separated and q u a n t i t a t e d . Leaf d i s c s or s e c t i o n s a l s o may be used to generate s u f f i c i e n t q u a n t i t i e s of metabolites f o r p u r i f i c a t i o n and chemical c h a r a c t e r i z a t i o n . However, use o f l e a f d i s c s or s e c t i o n s f o r such a purpose has been l i m i t e d . The i d e n t i f i c a t i o n o f g l u t a t h i o n e conjugation as one o f the major pathways f o r h e r b i c i d e d e t o x i c a t i o n i n p l a n t s was made through the use of sorghum l e a f d i s c s and s e c t i o n s . A water-soluble metabolite of a t r a z i n e was detected as a major metabolite i n sorghum l e a i n h i b i t e d photosynthesi from a l a r g e - s c a l e treatment o f sorghum l e a f s e c t i o n s bathed i n a s o l u t i o n o f a t r a z i n e f o r 20 hours (46). The metabolites, S-(4-ethylamino-6-isopropylamino-^-triaziny1-2)glutathione ( I I I ) and γ-glutamyl-jv- (4-ethylamino-6-isopropylamino-s_-triazinyl2 ) c y s t e i n e (IV), were s u c c e s s f u l l y i s o l a t e d and c h a r a c t e r i z e d from sorghum l e a f s e c t i o n s (46). Subsequent s t u d i e s with whole p l a n t s e l u c i d a t e d the mercapturic a c i d - l i k e pathway f o r a t r a z i n e metabolism i n p l a n t s (65). The l i m i t e d periods that l e a f d i s c s or s e c t i o n s may be allowed to metabolize x e n o b i o t i c s could be a disadvantage. Metabolites that a r e formed over longer periods cannot be generated i n l e a f d i s c s or s e c t i o n s . The metabolite of a t r a z i n e , N-(4-ethylamino-6-isopropylamino-s-triazinyl-2)lanthionine (VII), was present i n s i g n i f i c a n t q u a n t i t i e s i n leaves o f r o o t t r e a t e d sorghum p l a n t s only a f t e r 5 days (65). M e t a b o l i t e s I I I and IV were major metabolites i n sorghum shoots of whole p l a n t s a f t e r 14.4 hours o f treatment but d e c l i n e d t h e r e a f t e r (65). Therefore, due to temporal requirements i t i s d o u b t f u l i f metabolites such as V I I can be s u c c e s s f u l l y generated and i s o l a t e d from l e a f d i s c s or s e c t i o n s . Separated Leaf C e l l s . In t h i s method the mesophyll and p a l i s a d e c e l l s from leaves a r e separated to give a homo­ genous l i q u i d suspension of c e l l s that can be manipulated much l i k e u n i c e l l u l a r algae. The separated c e l l system i s u s e f u l f o r studying biochemical, p h y s i o l o g i c a l and cytochemical pro­ cesses i n p l a n t s . Use of i n d i v i d u a l c e l l s i n suspension permits equal exposure o f a l l c e l l s to t e s t chemicals. This i s u n l i k e the techniques i n v o l v i n g l e a f d i s c s o r s e c t i o n s i n which c e l l s along the cut edges a r e exposed p r e f e r e n t i a l l y to the t e s t chemicals. The presence o f r i g i d c e l l w a l l s permits repeated washing and c e n t r i f u g a t i o n of c e l l s with minimal damage. The metabolic f u n c t i o n o f the l e a f t i s s u e i s maintained i n the c e l l suspension but the s t r u c t u r a l i n t e g r i t y of the l e a f and the

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

24

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M E T A B O L I S M

normal f u n c t i o n of v a s c u l a r transport are no longer r e t a i n e d . Method. M o r p h o l o g i c a l l y i n t a c t mesophyll c e l l s may be separated by: 1) gently g r i n d i n g l e a f t i s s u e and separating i n d i v i d u a l c e l l s from l e a f d e b r i s by s e l e c t i v e f i l t r a t i o n and c e n t r i f u g a t i o n (66, 67); and 2) d i g e s t i o n of l e a f t i s s u e with enzyme preparations c o n t a i n i n g polygalacturonase and c e l l u l a s e (68, 69, 70, 71, 72). The enzyme s e p a r a t i o n methods p r e s e n t l y used are b a s i c a l l y m o d i f i c a t i o n s of those developed by Takebe et a l . (68) and Jensen et a l . (69). Separated l e a f c e l l s are prepared by c u t t i n g leaves i n t o s t r i p s (69) or squares (72) and vacuum i n f i l t r a t i n g these t i s s u e s with a maceration medium c o n t a i n i n g the enzyme (0.5 to 3.0%), i n o r g a n i c s a l t s b u f f e r (pH 5.8) potassium dextran s u l f a t e , a hypertonic s o l u t i o anti-senescent agents (2,4-D The i n f i l t r a t e d t i s s u e i s digested with a d d i t i o n a l maceration medium f o r 10 to 30 min. The c e l l s separated during t h i s f i r s t and a second s i m i l a r maceration periods are discarded. The c e l l s obtained during the t h i r d maceration p e r i o d (30 to 45 min) are most a c t i v e m e t a b o l i c a l l y and are used f o r experimen­ t a t i o n . The wash and assay media c o n t a i n s i m i l a r s a l t s as the maceration medium, 0.6 to 0.7 M s o r b i t o l and are b u f f e r e d at a higher pH (6.7 to 7.2). Discussion. The d i s c u s s i o n i s l i m i t e d to enzymatically separated c e l l s s i n c e t h i s i s the system that has been used to study the e f f e c t s of x e n o b i o t i c s ( h e r b i c i d e s ) on separated plant c e l l s . Maceration of l e a f t i s s u e i n a hypertonic environment that r e s u l t s i n c e l l p l a s m o l y s i s was necessary to y i e l d photos y n t h e t i c a l l y a c t i v e c e l l s (69). Plasmolyzed c e l l s with i n t a c t plasmalemmas were detected by phase c o n t r a s t microscopy (69). Separated cotton mesophyll c e l l s f i x e d C0£ at l i n e a r r a t e s of 50 to 100 ymoles/mg c h l o r o p h y l l /hour f o r 4 to 8 hours (71). Uptake of l ^ C - l e u c i n e and l ^ C - u r a c i l and i n c o r p o r a t i o n i n t o p r o t e i n and RNA, r e s p e c t i v e l y , occurred i n separated tobacco (Nicotiana tabacum L.) c e l l s (73). Uptake and i n c o r p o r a t i o n of the percursors i n t o macromolecules r e q u i r e d l i g h t and a c t i v e photosynthesis. A d d i t i o n of ATP only p a r t i a l l y s u b s t i t u t e d f o r the l i g h t requirement. A smaller percentage of the absorbed percursors was incorporated i n t o macromolecules when c e l l s were incubated i n the dark than i n l i g h t (73). E f f l u x of photosyn­ t h e t i c products amounted to 1 to 2% of t o t a l carbon f i x e d per hour (71). E f f l u x was i n h i b i t e d by the a d d i t i o n of Ca + i n the i n c u b a t i o n medium. The metabolic a c t i v i t y i s s i m i l a r between separated c e l l s and c e l l s of i n t a c t leaves and l e a f d i s c s described e a r l i e r . The separated c e l l s were m e t a b o l i c a l l y a c t i v e f o r 20 to 30 hours (69, 73). However, c r i t i c a l experimental periods should 2

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

SHiMABUKURO

A N D W A L S H

Plant Tissues, Organs, and

Isolated Cells

25

not exceed 5 to 12 hours s i n c e the metabolic a c t i v i t y i n the c e l l s may not be sustained beyond t h i s p e r i o d . Metabolism i n separated c e l l s . Separated c e l l s have been used to study p r i m a r i l y the e f f e c t s of h e r b i c i d e s and s u r f a c tants on plant c e l l membranes (74, 75, 76) and s e l e c t e d metab o l i c r e a c t i o n s (70, 72, 77, 78, 79). The biochemical and p h y s i o l o g i c a l changes observed i n the above s t u d i e s were not c o r r e l a t e d with metabolism of the x e n o b i o t i c s i n separated c e l l s . Therefore, the s t u d i e s are not complete. However, the p o t e n t i a l usefulness of t h i s technique has been demonstrated i n the l i m i t e d number of r e p o r t s . C a t i o n i c s u r f a c t a n t s increased s i g n i f i c a n t l y the e f f l u x of 14çQ2 f i x a t i o n products i n separated soybean w i l d onion (Allium canadense L.) an In w i l d onion, CO2 f i x a t i o s u r f a c t a n t s . Nonionic s u r f a c t a n t s had r e l a t i v e l y l e s s e f f e c t on membrane p e r m e a b i l i t y and photosynthesis than the c a t i o n i c s u r f a c t a n t s (74, 75). Wild onion c e l l s were l e s s s e n s i t i v e to the a c t i o n of a c a t i o n i c s u r f a c t a n t than were soybean c e l l s (75). Mixtures of o r y z a l i n O ^ - d i n i t r o - N ^ ^ - d i p r o p y l s u l f a n ilamide) with s e l e c t e d s u r f a c t a n t s enhanced e f f l u x i n soybean c e l l s above that of c e l l s t r e a t e d with o r y z a l i n or s u r f a c t a n t alone (74). O r y z a l i n and s u r f a c t a n t s caused greater e f f l u x of i n t r a c e l l u l a r m a t e r i a l from cotton c e l l s than from soybean c e l l s (74). The s y n e r g i s t i c e f f e c t s between o r y z a l i n and surf a c t a n t s were not observed i n cotton as with soybean c e l l s . The d i f f e r e n c e s observed between c e l l s from d i f f e r e n t species i n response to treatment with a chemical may not be due to inherent d i f f e r e n c e s i n t h e i r membranes. Such d i f f e r e n c e s may be only a r e f l e c t i o n of the d i f f e r e n c e s i n metabolism and d e t o x i c a t i o n of the b i o l o g i c a l l y a c t i v e compound between c e l l s from d i f f e r e n t s p e c i e s . Nonionic s u r f a c t a n t s l i k e T r i t o n X-100 are r a p i d l y metabolized i n p l a n t s when taken up by excised leaves or absorbed from l e a f surfaces (25, 80). The concept that h e r b i c i d e s have m u l t i p l e s i t e s of a c t i o n rather than a s i n g l e s i t e has been supported by r e s u l t s from separated c e l l s (72, 77, 78). A time-course e f f e c t of 13 major h e r b i c i d e s on photosynthesis, r e s p i r a t i o n , p r o t e i n s y n t h e s i s , RNA s y n t h e s i s , and l i p i d synthesis was measured i n separated kidney bean c e l l s a t v a r i o u s concentrations (72). Photosynt h e t i c i n h i b i t o r s such as a t r a z i n e , monuron, bromacil (5-bromo3-sec-butyl-6-methyluracil), and dinoseb (2-sec-butyl-4,6d i n i t r o p h e n o l ) i n h i b i t e d photosynthesis s i g n i f i c a n t l y but stimulated l i p i d synthesis at p h y s i o l o g i c a l concentrations of 0.1 to 1.0 μΜ. RNA synthesis a l s o was i n h i b i t e d at the same concentrations. At 100 μΜ, the same h e r b i c i d e s s t r o n g l y i n ­ hibited l i p i d synthesis. The i n h i b i t i o n of macromolecule s y n t h e s i s by photosynthetic i n h i b i t o r s i s probably a secondary e f f e c t s i n c e the uptake and i n c o r p o r a t i o n of percursors r e q u i r e

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

26

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M E T A B O L I S M

a c t i v e photosynthesis (73). The p h y s i o l o g i c a l s i g n i f i c a n c e of the reported e f f e c t s on metabolic processes may be evaluated b e t t e r i f information on the metabolism and i n t r a c e l l u l a r l o c a l i z a t i o n of the h e r b i c i d e s were known. The metabolism of d i f f e r e n t i a l l y l a b e l e d [ c ] f l u o r o d i f e n i n separated mesophyll c e l l s from r e s i s t a n t peanut i s shown i n Table I I I . The c e l l s were prepared using the method of Jensen et a l . (69) except that P e c t i n o l 41-P (crude pectinase and c e l l u l a s e ) was used i n p l a c e of macerozyme, no 2,4-D was added to any media, K2SO4 was s u b s t i t u t e d f o r potassium dextran s u l f a t e and the assay medium contained 0.6 M s o r b i t o l b u f f e r e d w i t h 0.05 M HEPES (pH 7.0). Assays were conducted i n 2 ml photosynthetic medium (69) that contained 10 μΜ [14c]fluorod i f e n , 1% acetone, 7.5 mM NaHC03 and separated c e l l s (238 μg c h l o r o p h y l l per r e a c t i o bated i n a d i f f e r e n t i a from below with incandescent lamps (5000 l u x ) . The c e l l s were separated from the assay medium and extracted with 80% methanol. The c e l l e x t r a c t and the assay medium were analyzed f o r f l u o r o d i f e n and i t s metabolites as reported p r e v i o u s l y (22, 81, 82). The uptake of f l u o r o d i f e n by peanut mesophyll c e l l s occurred r e a d i l y i n l i g h t or dark (Table I I I ) . The r a t i o of 14 i n the c e l l p e l l e t to that i n assay medium decreased with decreasing c e l l concentrations i n repeated experiments. Maxi­ mum uptake and metabolism of f l u o r o d i f e n occurred w i t h i n 2 hours. L i g h t appeared to have no i n f l u e n c e on the uptake of f l u o r o d i f e n . However, f l u o r o d i f e n metabolism was enhanced by l i g h t i n the 2-hour treatment p e r i o d . E f f l u x of water-soluble metabolites (glucosides and peptide conjugates) occurred from separated c e l l s . However, e f f l u x of f l u o r o d i f e n could not be determined. The r o l e of l i g h t i n the enhanced metabolism of f l u o r o d i f e n i s not c l e a r . Oxygen e v o l u t i o n was not i n h i b i t e d by f l u o r o d i f e n and ATP was not a r e q u i r e d c o - f a c t o r f o r g l u t a ­ thione S-transferase, the enzyme that c a t a l y z e s the ether cleavage of f l u o r o d i f e n (83). Separated z i n i a ( Z i n i a elegans Jacq.) c e l l s absorbed [14c]fluorodifen and [ 1 4 c ] t r i f l u r a l i n (α,α,α-trifluoro-2,6dinitro-N,N-dipropyl-£-toluidine) w i t h i n 2 hours (70). A f t e r washing c e l l s with s o l u t i o n s of the unlabeled h e r b i c i d e s , [ l ^ c j t r i f l u r a l i n appeared to be bound more s t r o n g l y than [ l ^ C ] f l u o r o d i f e n . However, the s i g n i f i c a n c e of the r e s u l t s i s not c l e a r s i n c e metabolism of the two compounds was not deter­ mined . The s i n g l e example of x e n o b i o t i c metabolism i n a separated c e l l system i l l u s t r a t e s the importance of metabolism when studying the mechanism of a c t i o n and s e l e c t i v i t y of a compound. Separated c e l l s can be used to measure r a p i d biochemical and p h y s i o l o g i c a l changes i n response to treatment with a xeno­ b i o t i c . However, the dynamic changes i n uptake, metabolism, 1 4

C

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

4

MeOH-insoluble residue:

3

1 100

58 9

31 1

dark

14

£

2

5 100

30 39

15 11

light

1 100

58 7

29 5

dark

4

2

/

[

27 40

16 10

3

CF ]

7 100

1 4

14

14

2

2

5 100

26 40

13 16

[ C-£-N0 -phenyl]

(light)

4-hour treatment

2.95 mCi/mmol; [ C-£-N0 -phenyl]

[ 4c- -N0 -phenyl]^

1

2-hour treatment

(%)

^/ [ C F 3 ] f l u o r o d i f e n : predominantly S7(2-nitro-4-trifluoromethylphenyl)gluthathione with small amounts of S-(2-nitro-4-trifluoromethylphenyl)-Nmalonylcysteine (82); [l C-£-N0 -phenyl]fluorodifen: mixture of £ nitrophenyl-3-D-glucoside and £-nitrophenyl-6-£-malonyl-$-D-glucoside (81).

1 4

2

D i s t r i b u t i o n of t o t a l l*c

Specific activities: [ CF3 f l u o r o d i f e n ] f l u o r o d i f e n - 2.7 mCi/mmol.

100

29 40

Cell Pellet: fluorodifen. conjugates—

light

CF3

[14 ]£/

18 9

Total

4

Metabolism of [ 1 C F ] - and [14c-£-N0 -phenyl]-labeled f l u o r o d i f e n i n separated peanut mesophyll c e l l s .

Assay medium: fluorodifen conjugates^/

Table I I I .

28

XENOBIOTIC

M E T A B O L I S M

and e f f l u x of the x e n o b i o t i c and i t s metabolites i n p l a n t c e l l s a l s o should be known before conclusions on mechanisms of a c t i o n and s e l e c t i v i t y are made. Plant P r o t o p l a s t s . P r o t o p l a s t s are naked c e l l s obtained by removal of t h e i r c e l l w a l l s . This may be accomp l i s h e d by: 1) m i c r o d i s s e c t i o n , or 2) enzymatic d i g e s t i o n . The l a t t e r i s the more e f f e c t i v e and commonly used procedure. The exposed plasmalemma d i f f e r e n t i a t e s p r o t o p l a s t s from i s o l a t e d mesophyll c e l l s whose r i g i d primary c e l l w a l l s are s t i l l i n t a c t . The same advantages described f o r i s o l a t e d mesophyll c e l l s apply to p r o t o p l a s t s . Because of the exposed plasmalemma, i t i s p o s s i b l e to study membrane absorption and p e r m e a b i l i t y e f f e c t s without i n t e r f e r e n c e by the c e l l w a l l (84) P r o t o p l a s t s have bee s t r u c t u r e and f u n c t i o n s on metabolism of x e n o b i o t i c s or t h e i r mechanisms of a c t i o n . This may be due to the d i f f i c u l t i e s of preparing and maintaining p r o t o p l a s t s ; they are extremely f r a g i l e and must be s t a b i l i z e d by osmotic s t a b i l i z e r s ( s o r b i t o l or mannitol) and C a salts (85, 86). 2 +

Method. Numerous methods f o r the i s o l a t i o n of plant p r o t o p l a s t s have been published, but no standard method e x i s t s s i n c e each s p e c i e s , type of t i s s u e , c e l l - s t r a i n , environmental growth c o n d i t i o n s , e t c . , r e q u i r e new adjustments i n the i s o l a t i o n procedures (85, 86). Generally, the methods are m o d i f i c a t i o n s of that o r i g i n a l l y published by Cocking (87). Each s p e c i a l problem appears to r e q u i r e e m p i r i c a l adjustments to a general scheme o u t l i n e d as follows (85): Leaves are s e l e c t e d from p l a n t s exposed to s p e c i f i c growth regimes and s u r f a c e s t e r i l i z e d . A l l operations are performed a s e p t i c a l l y . The lower epidermis of leaves i s removed to expose mesophyll c e l l s ; the leaves are cut i n t o s e c t i o n s and t r a n s f e r r e d to an enzyme s o l u t i o n . The enzyme s o l u t i o n f o r c e l l w a l l d i g e s t i o n may c o n t a i n pectinase and/or c e l l u l a s e and/or h e m i c e l l u l a s e with an osmotic s t a b i l i z e r and Ca2+. P r o t o p l a s t s , u s u a l l y r e l e a s e d w i t h i n 6 to 24 hours, are washed with a s o l u t i o n of the osmoticum and used f o r experimentation or c u l t u r e d under a s e p t i c c o n d i t i o n s f o r subsequent s t u d i e s . M o d i f i c a t i o n s to t h i s b a s i c procedure are i l l u s t r a t e d i n recent p u b l i c a t i o n s (88, 89, 90). Discussion. I s o l a t e d p l a n t p r o t o p l a s t s are g e n e r a l l y s p h e r i c a l i n appearance with c e l l u l a r components arranged along the c e l l periphery (91). P r o t o p l a s t s may undergo w a l l rejuven a t i o n , c e l l d i v i s i o n , and d i f f e r e n t i a t i o n to produce new p l a n t s under s u i t a b l e c o n d i t i o n s (86, 92). One of the greatest p o t e n t i a l s of p r o t o p l a s t s i s the production of i n t r a - and i n t e r - g e n e r i c hybrids through c e l l f u s i o n . Fusion of naked p r o t o p l a s t s from two tobacco species has been reported (93).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

SHIMABUKURO

A N D W A L S H

Phnt

Tissues, Organs, and

Isolated Cells

29

P r o t o p l a s t s may not be s u i t a b l e f o r l a r g e s c a l e gener­ a t i o n , p u r i f i c a t i o n , and c h a r a c t e r i z a t i o n of metabolites from x e n o b i o t i c s at present. However, i f the techniques f o r prepara­ t i o n of p r o t o p l a s t s are f u r t h e r improved and standardized, p r o t o p l a s t s may become another important t o o l f o r the p e s t i c i d e chemist. P r o t o p l a s t s may be u s e f u l f o r studying p h y s i o l o g i c a l and biochemical e f f e c t s of x e n o b i o t i c s i n plant c e l l s . Oat meso­ p h y l l p r o t o p l a s t s incorporated u r i d i n e and l e u c i n e up to 6 hours and thymidine up to 21 hours. K i n e t i n i n h i b i t e d l e u c i n e i n c o r p o r a t i o n with i n c r e a s i n g concentration of the growth hormone, but 2,4-D, a b s c i s i c a c i d and g i b b e r e l l i c a c i d had no e f f e c t on the same process (94). U r i d i n e i n c o r p o r a t i o n was i n h i b i t e d by 2,4-D at concentration abov 0.1 f the growth hormones had (94). The d i f f e r e n t i a l s u s c e p t i b i l i t y c e p t i b l e oat v a r i e t i e s to the phytotoxin, v i c t o r i n , was demonstrated with i s o l a t e d p r o t o p l a s t s (95). The c y t o k i n i n l i k e a c t i o n of methyl-2-benzimidazole carbamate (MBZ), the f u n g i t o x i c metabolite of benomyl [methyl-l-(butylcarbamoyl)-2benzimidazole carbamate], was confirmed i n oat p r o t o p l a s t s (96). The decrease i n nuclease a c t i v i t y , increase i n l e u c i n e i n c o r ­ p o r a t i o n and decrease i n u r i d i n e and thymidine i n c o r p o r a t i o n were s i m i l a r i n p r o t o p l a s t s t r e a t e d with k i n e t i n or MBZ (96). The e f f e c t of MBZ on the plasmalemma of p r o t o p l a s t s was not resolved. Tomato f r u i t p r o t o p l a s t s absorbed and r e t a i n e d s i g n i f i c a n t amounts of f l u o r o d i f e n and t r i f l u r a l i n (70). The short-term s t r u c t u r a l i n t e g r i t y of the plasmalemma from tomato p r o t o p l a s t s was unaffected by f l u o r o d i f e n , t r i f l u r a l i n , fluometuron [1,1dimethy1-3-(a,α,α-trifluoro-m-tolyl)urea], and chlorbromuron. However, complete membrane breakage and c o l l a p s e of the p r o t o ­ p l a s t s occurred a f t e r 30 minutes of treatment with paraquat ( l , l - d i m e t h y l - 4 , 4 - b y p y r i d i n i u m d i c h l o r i d e ) (97). The p r o t o p l a s t s have not been used to i n v e s t i g a t e metabolism of x e n o b i o t i c s . However, i t appears to be a convenient system to determine the metabolism of x e n o b i o t i c s concurrently with a study on the phytotoxic and s e l e c t i v e a c t i o n of these compounds i n plant c e l l s . ?

f

Conclusion. The survey of the l i t e r a t u r e on i n v i t r o organ, t i s s u e , and i s o l a t e d c e l l techniques f o r x e n o b i o t i c metabolism i n p l a n t s i s not complete. Selected r e p o r t s i n d i c a t e that i s o l a t e d plant systems may be used s u c c e s s f u l l y by p e s t i c i d e chemists to e l u c i d a t e degradation pathways and i n t e r a c t i o n s of x e n o b i o t i c compounds i n p l a n t s . I s o l a t e d p l a n t systems are most u s e f u l f o r short-term i n v e s t i g a t i o n s on metabolism, mechanism of a c t i o n and s e l e c t i v i t y .

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One of the major advantages of the techniques discussed in this report is the ability to measure rapid biochemical and physiological changes in response to a chemical coupled to its metabolism under carefully controlled conditions. This may not be possible in whole-plant experiments. Reduced growth and other visible manifestations of injury in whole plants are usually much delayed secondary responses to a chemical. How­ ever, results from isolated plant systems must be carefully evaluated before they are extrapolated to intact whole plants. Also, conclusions based only on whole-plant experiments should be confirmed by in vitro techniques. Abstract. Whole plants have and "terminal" residue using isolated leaves or roots, leaf discs or sections, and separated mesophyll cells have proven to be more useful for short-term investigations on xenobiotic metabolism, mechanism of action and selectivity. The in vitro techniques allow the treatment of large amounts of plant material with a minimum of chemical and facilitate rapid uptake of the chemical into plant tissues. The influence of root and leaf surface absorption is also circumvented by the use of in vitro techniques. Quan­ titative differences in xenobiotic metabolism between whole plants and isolated plant systems have been observed, but qualitative differences are not common. Results from isolated plant systems must be carefully evaluated before extrapolation to intact plants because of the unknown influence of senescence, absorption, and translocation on in vitro systems. Literature Cited 1. Casida, J. E. and Lykken, L., Annu. Rev. Plant Physiol. (1969) 20, 607. 2. Kearney, P. C. and Kaufman, D. D., eds., "Herbicides Chemistry, Degradation and Mode of Action," Vol. 1 and 2, Marcel Dekker, Inc., New York, NY (1975). 3. Frear, D. S., Hodgson, R. Η., Shimabukuro, R. H. and Still, G. G., Advan. Agron. (1972) 24, 328. 4. Bukovac, M. J., "Herbicides - Physiology, Biochemistry, Ecology," Audus, L. J., ed., Vol. 1, p. 335, Academic Press, New York, NY (1976). 5. Hay, J. R., "Herbicides - Physiology, Biochemistry, Ecology," Audus, L. J., ed., Vol. 1, p. 365, Academic Press, New York, NY (1976). 6. Haynes, R. J. and Goh, Κ. Μ., Scien. Hort. (1977) 7, 291. 7. Crafts, A. S. and Crisp, C. E., "Phloem Transport in Plants," W. H. Freeman and Co., San Francisco, CA (1971).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1. SHIMABUKURO AND WALSH Plant Tissues, Organs, and Isolated Cells 31 8. Zimmerman, M. H. and Milburn, J. Α., eds., "Transport in Plants I: Phloem Transport," Encyclopedia Plant Physiol. (N.S.), Vol. 1, Springer-Verlag, Heidelberg, West Germany (1975). 9. Crafts, A. S., in "The Physiology and Biochemistry of Herbicides," Audus, L. J., ed., p. 75, Academic Press, New York, NY (1964). 10. Woolhouse, H. W., Sci. Prog. (1974) 61, 123. 11. Thimann, Κ. V., "Hormone Action in the Whole Life of Plants," p. 357, University of Massachusetts Press, Amherst, MA (1977). 12. Goldthwaite, J. J. and Laetsch, W. Μ., Plant Physiol. (1967) 42, 1757. 13. Peterson, L. W. and Huffaker R C., Plant Physiol (1975) 55, 1009. 14. Tetley, R. M. and , , Physiol (1974) 54, 294. 15. Sodek, L. and Wright, S. T. C., Phytochem. (1969) 8, 1629. 16. Haber, Α. Η., Thompson, P. J., Walne, P. L. and Triplett, L. L., Plant Physiol. (1969) 44, 1619. 17. Martin, C. and Thimann, Κ. V., Plant Physiol. (1972) 49, 64. 18. Frear, D. S. and Swanson, H. R., Phytochem. (1972) 11, 1919. 19. Frear, D. S. and Swanson, H. R., Phytochem. (1974) 13, 357. 20. Frear, D. S. and Swanson, H. R., Pest. Biochem. Physiol. (1975) 5, 73. 21. Lamoureux, G. L. and Stafford, L. E., J. Agric. Food Chem. (1977) 25, 512. 22. Shimabukuro, R. Η., Lamoureux, G. L . , Swanson, H. R., Walsh, W. C., Stafford, L. E. and Frear, D. S., Pestic. Biochem. Physiol. (1973) 3, 483. 23. Lamoureux, G. L . , Stafford, L. E. and Shimabukuro, R. Η., J. Agric. Food Chem. (1972) 20, 1004. 24. Lamoureux, G. L., Stafford, L. E. and Tanaka, F. S., J. Agric. Food Chem. (1971) 19, 346. 25. Stolzenberg, G. E. and Olson, P. Α., 173rd Amer. Chem. Soc. Meeting (1977) Abst. Pest. 42. 26. Shimabukuro, R. H., Masteller, V. J. and Walsh, W. C., Weed Sci. (1976) 24, 336. 27. Hamilton, R. H., J. Agric. Food Chem. (1964) 12, 14. 28. Swanson, C. R., Kadunce, R. E., Hodgson, R. H. and Frear, D. S., Weeds (1966) 14, 319. 29. Shimabukuro, R. H., J. Agric. Food Chem. (1967) 15, 557. 30. Shimabukuro, R. Η., Walsh, W. C. and Hoerauf, R. Α., (unpublished data). 31. Shimabukuro, R. Η., Plant Physiol. (1967) 42, 1269. 32. Carter, M. C., "Herbicides - Chemistry, Degradation and Mode of Action," Kearney, P. C. and Kaufmann, D. D., eds., Vol. 1, p. 377, Marcel Dekker, Inc., New York, NY (1975) 33. Lund-Höie, Κ., Weed Res. (1970) 10, 367.

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34. Basler, Ε., Murray, D. S. and Santelmann, P. W., Weed Sci. (1978) 26, 358. 35. Hoagland, R. E. and Frear, D. S., J. Agric. Food Chem. (1976) 24, 129. 36. Shimabukuro, R. Η., Frear, D. S., Swanson, H. R. and Walsh, W. C., Plant Physiol. (1971) 47, 10. 37. Frear, D. S. and Swanson, H. R., Phytochem. (1970) 9, 2123. 38. S t i l l , G. G. and Mansager, E. R., Pestic. Biochem. Physiol. (1973) 3, 87. 39. Shtarkshall, R. Α., Reinhold, L. and Harel, H., J. Exp. Bot. (1970) 21, 915. 40. Nissen, P., Annu. Rev. Plant Physiol. (1974) 25, 53. 41. Morrod, R. S., J. Exp. Bot. (1974) 25, 521. 42. Jensen, Κ. I. Ν. Banden J D. and SouzaMachado V. Can. J. Plant Sci 43. Miflin, B. J., Plant 44. Shimabukuro, R. H. and Swanson, H. R., J. Agric. Food Chem. (1969) 17, 199. 45. Lien, R. and Rognes, S. Ε., Physiol. Plant. (1977) 41, 175. 46. Lamoureux, G. L . , Shimabukuro, R. Η., Swanson, H. R. and Frear, D. S., J. Agric. Food Chem. (1970) 18, 81. 47. Jones, H. G., Aust. J. Biol. Sci. (1973) 26, 25. 48. Jensen, Κ. I. Ν., Stephenson, G. R. and Hunt, L. Α., Weed Sci. (1977) 25, 212. 49. MacDonald, I. R., Plant Physiol. (1975) 56, 109. 50. Chollet, R., Plant Physiol. (1978) 61, 929. 51. Morrod, R. S., "Herbicides - Physiology, Biochemistry, Ecology," Audus, L. J., ed., Vol. 1, p. 281, Academic Press, New York, NY (1976). 52. Cheung, Y. K. S. and Nobel, P. S., Plant Physiol. (1973) 52, 633. 53. Donaldson, T. W., Bayer, D. E. and Leonard, O. Α., Plant Physiol. (1973) 52, 638. 54. Swanson, C. R. and Swanson, H. R., Weed Sci. (1968) 16, 137. 55. Swanson, C. R. and Swanson, H. R., Weed Sci. (1968) 16, 481. 56. Shimabukuro, R. Η., Swanson, H. R. and Walsh, W. C., Plant Physiol. (1970) 46, 103. 57. Bowling, C. C. and Hudgins, H. R., Weeds (1966) 14, 94. 58. Matsunaka, S., Science (1968) 160, 1360. 59. Frear, D. S. and Still, G. G., Phytochem. (1968) 7, 913. 60. Chang, F-Y., Smith, L. W. and Stephenson, G. R., J. Agric. Food Chem. (1971) 19, 1183. 61. Chang, F-Y., Stephenson, G. R. and Smith, L. W., J. Agric. Food Chem. (1971) 19, 1187. 62. Hamill, A. S. and Penner, D., Weed Sci. (1973) 21, 330. 63. Hamill, A. S. and Penner, D., Weed Sci. (1973) 21, 335. 64. Hamill, A. S. and Penner, D., Weed Sci. (1973) 21, 339.

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65. Lamoureux, G. L . , Stafford, L. Ε., Shimabukuro, R. H. and Zaylskie, R. G., J. Agric. Food Chem. (1973) 21, 1020. 66. Gnanam, A. and Kulandaivelu, G., Plant Physiol. (1969) 44, 1451. 67. Edwards, G. E. and Black, C. C. Jr., Plant Physiol. (1971) 47, 149. 68. Takebe, I., Otsuki, Y. and Aoki, S., Plant Cell Physiol. (1968) 9, 115. 69. Jensen, R. G., Francki, R. I. B. and Zaitlin, Μ., Plant Physiol. (1971) 48, 9. 70. Boulware, M. A. and Camper, N. D., Weed Sci. (1973) 21, 145. 71. Rehfeld, D. W. and Jensen, R. G., Plant Physiol. (1973) 52, 17. 72. Ashton, F. Μ., DeVilliers W. B., Pestic. Biochem Physiol (1977) 7, 73. Francki, R. I. B., Zaitlin, M. and Jenson, R. G., Plant Physiol. (1971) 48, 14. 74. Towne, C. Α., Bartels, P. G. and Hilton, J. L . , Weed Sci. (1978) 26, 182. 75. St. John, J. B., Bartels, P. G. and Hilton, J. L . , Weed Sci. (1974) 22, 233. 76. Davis, D. G. and Shimabukuro, R. Η., Weed Sci. Soc. Abst. No. 157 (1973). 77. Porter, Ε. M. and Bartels, P. G., Weed Sci. (1977) 25, 60. 78. Radosevich, S. R. and DeVilliers, O. T., Weed Sci. (1976) 24, 229. 79. DeVilliers, O. T. and Ashton, F. Μ., Agroplantae (1976) 8., 87. 80. Stolzenberg, G. E. and Olson, P. Α., 175th Amer. Chem. Soc. Meeting (1978) Abst. Pest. 67. 81. Shimabukuro, R. Η., Walsh, W. C., Stolzenberg, G. E. and Olson, P. Α., Weed Sci. Soc. Am. (1975) Abst. No. 171. 82. Shimabukuro, R. Η., Walsh, W. C., Stolzenberg, G. E. and Olson, P. Α., Weed Sci. Soc. Am. (1976) Abst. No. 196. 83. Frear, D. S. and Swanson, H. R., Pestic. Biochem. Physiol. (1973) 3, 473. 84. Ruesink, A. W., Plant Physiol. (1971) 47, 192. 85. Constabel, F., in "Plant Tissue Culture Methods," Gamborg, O. L. and Wetter, L. R., eds., p. 11, Prairie Regional Laboratory, Saskatoon, Canada (1975). 86. Cocking, E. C., Annu. Rev. Plant Physiol. (1972) 23, 29. 87. Cocking, E. C., Nature (1960) 187, 962. 88. Farmer, I. and Lee, P. Ε., Plant Sci. Lett. (1977) 10, 141. 89. Cassells, A. C. and Barlass, Μ., Physiol. Plant. (1978) 42, 236. 90. Watts, J. W., Motoyoshi, F. and King, J. Μ., Ann. Bot. (1974) 38, 667. 91. Takebe, I., Otsuki, Υ., Honda, Y., Nishio, T. and Matsui,

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Gamborg, O. L. and Miller, R. Α., Can. J . Bot. (1973) 51, 1795. Carlson, P. S., Smith, H. H. and Dearing, R. D., Proc. N a t l . Acad. S c i . U.S.A. (1972) 69, 2292. Fuchs, Y. and Galston, A. W., Plant C e l l P h y s i o l . (1976) 17, 475. R a n c i l l a c , M., Kaur-Sawhney, R., Staskawicz, B. and Galston, A. W., Plant C e l l P h y s i o l . (1976) 17, 987. Staskawicz, B., Kaur-Sawhney, R., Slaybaugh, R., Adams, W. J r . and Galston, A. W., P e s t i c . Biochem. P h y s i o l . (1978) 8, 106. Boulware, M. A. and Camper, D. Ν., P h y s i o l . P l a n t . (1972) 26, 313. December 20,

1978

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2 Xenobiotic Metabolism in Higher Plants: In Vitro Tissue and Cell Culture Techniques RALPH O. MUMMA and ROBERT H. HAMILTON Pesticide Research Laboratory and Graduate Study Center and Departments of Entomology and Biology, Pennsylvania State University, University Park, PA 16802 M i l l i o n s of pounds of x e n o b i o t i c s have been a p p l i e d to p l a n t s in our environment f o r th c o n t r o l f pest d p l a n t growth Some of these chemicals b i o l o g i c a l e f f e c t s on animal plants, , such as o i l s , adjuvants, e m u l s i f i e r s and i n e r t m a t e r i a l s , are a p p l i e d i n even greater q u a n t i t y , but have been assumed to cause l i t t l e ecological effect. I t i s important to understand what happens to a l l chemicals a p p l i e d t o our environment and to p r o p e r l y i n t e r p r e t the e c o l o g i c a l s i g n i f i c a n c e of these chemicals and of t h e i r degradation products. I f we cannot know t h i s i n d e t a i l , then a general knowledge of p e r s i s t a n c e and metabolic products i s of importance. Since the t a r g e t organism of these x e n o b i o t i c s i s o f t e n p l a n t s , i t i s of the utmost importance to understand the f a t e of these chemicals i n p l a n t s . U l t i m a t e l y , animals and even humans are exposed to these chemicals and/or t h e i r subsequent metabolites and degradation products. Most i n v e s t i g a t i o n s of x e n o b i o t i c metabolism i n p l a n t s have focused on b i o l o g i c a l l y a c t i v e pest c o n t r o l chemicals. Thus, t h i s review w i l l a l s o focus p r i m a r i l y on p l a n t metabolism of p e s t i c i d e s . There are many ways to study the metabolism of x e n o b i o t i c s by p l a n t s , but whatever technique i s employed, i t should p r e d i c t what would a c t u a l l y happen under f i e l d c o n d i t i o n s . Metabolism s t u d i e s have i n v o l v e d whole p l a n t s , excised p l a n t p a r t s (meristems, shoots, stems, l e a v e s , r o o t s , l e a f d i s k s ) , p l a n t c e l l c u l t u r e s , s u b c e l l u l a r p a r t i c l e s , and i s o l a t e d enzymes. Any metabolism study conducted i n the l a b o r a t o r y i s l e s s than i d e a l because i t i s d i f f i c u l t to d u p l i c a t e many f a c t o r s that a f f e c t the degradation of x e n o b i o t i c s under f i e l d c o n d i t i o n s such as weather, l i g h t , microsymbionts, s o i l or method of a p p l i c a t i o n . Metabolism of xenobiot i c s by p l a n t t i s s u e c u l t u r e o f f e r s the obvious advantages of s t e r i l i t y , space, economical use of l a b e l e d chemicals, l e s s p i g ments, e t c . These advantages w i l l be discussed i n d e t a i l l a t e r . The metabolism of x e n o b i o t i c s by p l a n t t i s s u e c u l t u r e (1) and the metabolism of endogenous and exogenous chemicals (2) have been reviewed r e c e n t l y . W i t h i n the l a s t few y e a r s , many new

0-8412-0486-l/79/47-097-035$10.50/0 © 1979 American Chemical Society

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i n v e s t i g a t i o n s of x e n o b i o t i c metabolism by p l a n t t i s s u e c u l t u r e s have been reported. This review w i l l focus on these more recent papers with an emphasis on p e s t i c i d e metabolism. We w i l l attempt to evaluate and compare the r e s u l t s obtained w i t h t i s s u e c u l t u r e techniques versus those obtained w i t h whole p l a n t s and to explore the many f a c t o r s ( l i m i t a t i o n s ) that a f f e c t metabolism s t u d i e s with plant tissue culture. H i s t o r y and P r i n c i p l e s of P l a n t T i s s u e Culture Plant t i s s u e c u l t u r e r e f e r s to the growth of r e l a t i v e l y und i f f e r e n t i a t e d p l a n t c e l l s or d i f f e r e n t i a t e d organs on s o l i d or l i q u i d n u t r i e n t medium. The u n d i f f e r e n t i a t e d p l a n t t i s s u e growing on s o l i d i f i e d medium i s u s u a l l y r e f e r r e d to as a c a l l u s c u l t u r e s i n c e they a r e f r e q u e n t l and maintain the appearanc t i s s u e s are placed i n l i q u i d medium w i t h shaking, many small aggregates of c e l l s and even some s i n g l e c e l l s may be obtained. Subcultures of small aggregates or c e l l clumps i n l i q u i d c u l t u r e are u s u a l l y designated as suspension c u l t u r e s . The c u l t u r e of excised d i f f e r e n t i a t e d organs i s , of course, organ c u l t u r e . F r e quently, c a l l u s c u l t u r e s w i l l d i f f e r e n t i a t e w i t h the formation of xylem elements and sometimes buds and/or r o o t s . In many c a l l u s c u l t u r e s with t h i s p o t e n t i a l u s u a l l y a high k i n e t i n / a u x i n concent r a t i o n r a t i o i n the medium favors bud formation while the reverse favors root formation. In a few cases, by proper manipulation of the medium, thousands of pseudoembryos can be induced and grown i n t o normal p l a n t s of the same genotype. Although the c u l t u r e of p l a n t t i s s u e s was attempted i n 1902 (3), success was not achieved u n t i l White c u l t u r e d excised tomato roots i n 1934 and tobacco c a l l u s i n 1939 (4). Gautheret and Nobêcourt a l s o described c u l t u r e of p l a n t c a l l u s t i s s u e at about the same time (5, 6) . White and Gautheret both developed n u t r i e n t media that a r e used widely today (5> 2) · In a d d i t i o n to e s s e n t i a l s a l t s and sucrose, White added thiamine, n i c o t i n i c a c i d , pyridoxine and g l y c i n e while Gautheret added thiamine, pantothenic a c i d , b i o t i n , i n o s i t o l and c y s t e i n e . I t appears that only thiamine i s e s s e n t i a l ; but, i n some cases, b e t t e r growth may be obtained by the a d d i t i o n of other v i t a m i n s . White d i d not add an auxin f o r c u l t u r e of excised root organ c u l t u r e s or tobacco tumor t i s s u e , but Gautheret added naphthaleneacetic a c i d (NAA). Van Overbeek et a l . (8) introduced the use of coconut milk and much l a t e r M i l l e r et a l . (9) found that k i n e t i n was necessary to c u l t u r e tobacco stem p i t h . In g e n e r a l , excised root organ c u l t u r e s , tumor and some c a l l u s c u l t u r e s (so c a l l e d habituated) do not r e q u i r e e i t h e r auxin or k i n e t i n . Some t i s s u e s do not r e q u i r e k i n e t i n e s p e c i a l l y i f 2,4-dichlorophenoxyacetic a c i d (2,4-D) i s used as the auxin (10). The a d d i t i o n of coconut milk i s u s u a l l y not e s s e n t i a l , and a requirement f o r g i b b e r e l l i c a c i d has been reported r a r e l y . I t i s probable that adaptation to the medium

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Cultures

37

and the s e l e c t i o n of t i s s u e that grows w e l l on a p a r t i c u l a r medium accounts f o r the wide v a r i a t i o n i n n u t r i e n t requirements f o r a t i s s u e such as tobacco c a l l u s . Other media used widely f o r plant t i s s u e c u l t u r e i n c l u d e those of Murashige and Skoog (11), N i t s c h and N i t s c h (12) and Gamborg (13). In a d d i t i o n , some media formul a t i o n s are now a v a i l a b l e from a commercial source (Flow Laborat o r i e s , P. 0. Box 2226, 1710 Chapman Ave., R o c k v i l l e , MD 20852). In theory c a l l u s may be derived from any p l a n t t i s s u e cont a i n i n g parenchyma c e l l s . Some species form c a l l u s r e a d i l y and others do not. Tissue s t e r i l i z a t i o n may be accomplished with 70% a l c o h o l (1-2 min dip) and/or a s i m i l a r treatment with 1 to 5 or 1 to 10 d i l u t e d commercial bleach (0.5-1% sodium hypochlor i t e ) containing 0.1% Tween 20. In e i t h e r case, the t i s s u e i s r i n s e d 2-3 times i n s t e r i l e water. Seeds are germinated i n s t e r i l e p e t r i dishes and b i t to s o l i d i f i e d agar mediu (0.5-1.0 mg/1 NAA or 2,4-D). S t e r i l i z e d t i s s u e from bud, leaves or stems may a l s o be used. Formation of enough c a l l u s to s u b c u l ture may vary from 2 weeks to 2 months. Considerable v a r i a t i o n i n appearance and texture between c a l l u s pieces from the same source may be observed. Three to 4 b i t s of c a l l u s (5-7 mm i n dia.) are t r a n s f e r r e d to s o l i d i f i e d agar medium (50 ml i n a 125 ml Erlenmeyer f l a s k ) . D i f f i c u l t t i s s u e s may r e q u i r e the a d d i t i o n of 50 ml of a u t o c l a v e d - f i l t e r e d coconut l i q u i d and/or 2 g of c a s e i n hydrolysate per l i t e r . Growth rates vary, but i t i s convenient to subculture b i t s of c a l l u s onto f r e s h medium every 4^6 weeks. Temperature and l i g h t requirements may not be c r i t i c a l f o r most t i s s u e s . High temperatures (30°C or more) have caused the l o s s of k i n e t i n dependence of tobacco c a l l u s (14) and i n h i b i t e d growth. We have maintained c u l t u r e s under continuous low l e v e l f l u o r e s c e n t l i g h t at 27°C. Simple equipment i s needed f o r p l a n t t i s s u e c u l t u r e s ; a temperature c o n t r o l l e d incubator or c u l t u r e room, an autoclave, and a shaker, i f suspension c u l t u r e s are grown. I t i s a l s o d e s i r a b l e to have a s t e r i l e t r a n s f e r hood. The genetic and p h y s i o l o g i c a l s t a b i l i t y of some plant t i s s u e c u l t u r e s i s of concern. T i s s u e from the same o r i g i n a l c u l t u r e may change i n appearance and growth r a t e over a period of time. It i s commonly observed that the a b i l i t y to regenerate normal p l a n t s by the formation of buds and roots i s l o s t with time. I t may even be necessary to r e i s o l a t e c a l l u s from the same source, i f the c a l l u s t i s s u e appears a t y p i c a l or slow growing. A low growth r a t e i s observed f r e q u e n t l y i n August or September. V a r i ations i n growth r a t e and t i s s u e appearance may be important f a c t o r s i n metabolism studies and should be examined c a r e f u l l y . A m i t o t i c index at two weeks a f t e r t r a n s f e r may give a good i n d i c a t i o n of growth r a t e (15) or f r e s h weights at the end of 4 or 5 weeks may be used. The source of c a l l u s t i s s u e (root, stem, l e a f , cotyledons, etc.) a l s o may i n f l u e n c e metabolism. The appearance of soybean cotyledon, l e a f and root c a l l u s was s i m i l a r and l i m i t e d

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

38

XENOBIOTIC

M E T A B O L I S M

work on 2,4-D metabolism i n d i c a t e d no major q u a l i t a t i v e d i f f e r ­ ences (16). Of much more importance was the age or stage of grow­ th of the c u l t u r e s (15). Most c a l l u s c u l t u r e s e x h i b i t a l a g i n growth f o r 4-7 days a f t e r t r a n s f e r , a l o g growth phase followed by a slower growth phase a f t e r 4-5 weeks. Cultures 5-9 weeks o l d are more a c t i v e i n metabolism of 2,4-D than 3 week o l d c u l t u r e s (15). I t should be noted that prolonged p e s t i c i d e treatment of o l d e r c a l l u s i n f r e s h medium may put the t i s s u e i n t o l o g phase growth again w i t h probable changes i n metabolism. Metabolism of Xenobiotics

by Plant Tissue

Culture

Most i n v e s t i g a t i o n s w i t h p l a n t t i s s u e c u l t u r e s and xenobiot­ i c s have been concerned w i t h p e s t i c i d e metabolism. Tables I and I I i l l u s t r a t e the v a r i e t t i s s u e c u l t u r e s that hav metabolism of lindane probably represents one of the more extreme cases where fourteen d i f f e r e n t p l a n t t i s s u e s were used. As i s evident, the source of the p l a n t t i s s u e c u l t u r e , the type of c u l ­ ture (suspension or nonsuspension) and the media used a l s o v a r i e d . This v a r i a t i o n i n p l a n t c u l t u r e s and techniques makes i t extremely d i f f i c u l t to c r i t i c a l l y compare metabolism s t u d i e s . As w i l l be pointed out l a t e r , there are a l s o many other f a c t o r s that a f f e c t metabolism s t u d i e s with p l a n t t i s s u e c u l t u r e s . HERBICIDES I t i s apparent that h e r b i c i d e s exert t o x i c or p h y s i o l o g i c a l e f f e c t s on s e n s i t i v e plant species and that some p h y s i o l o g i c a l e f f e c t s can be expected on t i s s u e c u l t u r e s of these species. An exception may be photosynthetic i n h i b i t o r s . Due to the sugar i n the p l a n t t i s s u e c u l t u r e medium, photosynthetic i n h i b i t o r s should not be s t r o n g l y i n h i b i t o r y unless they have secondary s i t e s of action. I f t o l e r a n c e i s due to metabolism, t h i s might be expected to lead to q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s i n metabolism by p l a n t t i s s u e c u l t u r e s from s u s c e p t i b l e and r e s i s t a n t v a r i e t i e s or s p e c i e s . Of course, such t i s s u e c u l t u r e s should a l s o show differences i n actual tolerance. The metabolism of the h e r b i c i d e f l u o r o d i f e n (p-nitrophenyl α,α,a-trifluoro-2-nitro-p-tolyl ether) has been i n v e s t i g a t e d (21) with tobacco c e l l s i n suspension c u l t u r e (42). The c e l l s were incubated f o r 15 days w i t h 1-2 ppm of e i t h e r C i - or CF3-labeled f l u o r o d i f e n . A l l of the a p p l i e d f l u o r o d i f e n was metabolized. Recovery of added r a d i o a c t i v i t y v a r i e d between 52 and 76%. The c e l l s contained 60 to 80% of the recovered r a d i o a c t i v i t y and the remainder was found i n the medium and c e l l wash. With ^Ci-label­ ed f l u o r o d i f e n , 4-nitrophenol (7%) was i s o l a t e d only from the medium. Aqueous-soluble conjugated forms of 4-nitrophenol (93%), p r i m a r i l y the 3-D-glucoside and other a c i d i c conjugates, were pre­ sent i n both the c e l l s and the medium as summarized i n Figure 1. l l f

1

lf

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3

2

ι H

>-N-c-cH cH

Propanil

\=J

CI- • f

0

Diphenimid

CI

^

3

v CH

ÇH3_

Ο

^ N-CCH^ CH ^ ^

Cisanilide

3

ι

0

3

Rice

1

(Oryza s a t i v a L. v a r . Starbonnet)

1

Leaves

Source

Root

Soybean (Glycine max [L.] Merr. Root t i p s Wilkin )

Carrot (Daucus c a r o t a L.) Cotton (Gossypium hirsutum L.)

Species

H, S

B5, S

B5,

Medium and ^ C u l t u r e Type

Plant T i s s u e C u l t u r e

Metabolism of H e r b i c i d e s by P l a n t Tissue C u l t u r e s .

Herbicide

Table I.

20

19

17, 18

Reference.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

3

Ν — Ν

Cl

0

NH

2,4-D

/Cl

2

OCH2COOH

Metribuzin

3

^-SCH

Fluorodifen

(CH ) C-/

N0

NO2

Table I - page 2

3

( C u l t i v a r Bragg) ( C u l t i v a r Coker 102)

Cotyledon Cotyledon

Soybean (Glycine max L. v a r Root Mandarin) Soybean (Glycine max L. v a r Cotyledon Acme) Jackbean (Canavalia ensiformis)Pod Endosperm Sweet corn (Zea mays) Tobacco (Nicotiana tobacum) Pith Carrot (Daucus c a r o t a v a r Pith Sativa) Sunflower (Helianthus annus) P i t h Root Rice (Oryza s a t i v a v a r Starbonnet) Wheat ( T r i t i c u m monococcum L. ) Stem F i e l d bindweed (Convolvulus a r v e n i s L.)

Soybean Soybean

Tobacco (Nicotiana tobacum L. var Xanthi)

24, 26, 27 27 27 27 28 29 30

C C C C

M, M, M, M,

M, C M, C B5, S X, C

23

22

21

M, C

B5

X, S X, S

M, S

W Ο Ε

"s

ο

i

w

X m ο

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. Ovular

C

MS,

W,

Bark

C C

W,

Crown-gall W,

Stem

M, S

C

S - Suspension; C - C a l l u s .

f

Geranium (Pelagonium hortorum var N i t t a n y Red) Boston i v y (Parthenocissus tricuspidata) Apple (East M a i l i n g r o o t s t o c k 3430) Shamouti orange ( C i t r u s s i n e n s i s Osb.

Cotyledon

C u l t u r e type:

,CH COOH

L. var

B5 - Gamborg's; H - H e l l e r ' s ; M - M i l l e r s ; MS - Murashige

IAA

2,4,5-T

CI

2

^ - 0 C H COOH

Soybean (Glycine max Acme)

B a s i c media: X - Others.

CI-

Table I - page 3

f

35

1

and Skoog s; W - W h i t e s

36

34,

33

32

31

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

O-C-NHCH3

Potato (Solanum tuberosum)

Purple cockle (Agrostemma githago L.) Soybean (Glycine max) Bedstraw (Galium verum) Carrot (Daucus carota) Clover (Meliotus alba) Tobacco ( N i c o t i a n a tobacum) Tobacco ( N i c o t i a n a g l u t i n o s a ) Tobacco ( N i c o t i a n a s y l v e s t r i s ) Tobacco ( N i c o t i a n a glauca) Lettuce (Lactuca s a t i v a ) (Beta v u l g a r i s ) P a r s l e y (Pstroselinum hortense)

Source

Culture

M,

S

Medium and ^ Culture Type

Plant Tissue

Cultures.

Tobacco ( N i c o t i a n a tobacum L. var Xanthi)

Species

Metabolism of I n s e c t i c i d e s by Plant Tissue

Insecticide

Table I I .

39

37,

38

Reference

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

i

f

3

x x

B a s i c media:

-Cl

-Cl

P a r s l e y (Petroselinum hortense, Hoffm.) Soybean (Glycine max L.)

P a r s l e y (Petroselinum hortense, Hoffm.) Soybean (Glycine max L.)

Bean (Phaseolus v u l g a r i s var Roots & Canadian Wonder) Shoots Potato (Solanum tuberosum var Tuber Majestic)

S

41 41 B5, S

41

40

40

B5, S

B5, S

W, S

MS,

S - Suspension; C - C a l l u s .

M - M i l l e r ' s ; W - White's; MS - Murashige and Skoog's; B5 - Gamborg's; X - Other.

Kelthane (Ascaroside)

CC1

OTO

OH

£,£ -DDT

CCI 3

~ 0 ~ K

^ C u l t u r e type:

a

Cl-

c

H

Table I I - page 2 Ql _ CL Cl'

44

XENOBIOTIC

M E T A B O L I S M

NO

roduct

(79% Journal of Agricultural and Food Chemistry Figure 1.

Metabolism of fluorodifen by tobacco cell in suspension culture

These data from plant t i s s u e c u l t u r e s t u d i e s are c o n s i s t a n t with r e s u l t s obtained w i t h whole p l a n t s where the glucoside of 4n i t r o p h e n o l has been reported as the major product of soybean and maize seedlings (43, 44). Whole p l a n t metabolism s t u d i e s with f l u o r o d i f e n (43, 44) suggested that a small percentage of the a p p l i e d p e s t i c i d e may be reduced to 4-aminophenyl 2-amino-4-(trifluoromethyl) phenyl ether, 4-nitrophenyl 2-amino-4-(trifluoromethyl) phenyl ether or 4-aminophenyl 2 - n i t r o - 4 - ( t r i f l u o r o m e t h y l ) phenyl ether, but none of these compounds, i n c l u d i n g 4-aminophenyl, was detected i n the tobacco t i s s u e c u l t u r e s t u d i e s . P r o p a n i l ( 3 , 4 - d i c h l o r o p r o p i o n a n i l i d e ) i s an h e r b i c i d e used to c o n t r o l weeds i n r i c e . The r e s i s t a n c e of r i c e to p r o p a n i l i s a t t r i b u t e d to the high l e v e l s of an a r y l a c y l amidase ( p r o p a n i l amidase) that hydrolyzes p r o p a n i l to 3 , 4 - d i c h l o r o a n i l i n e and prop i o n i c a c i d . The enzymatic l e v e l of p r o p a n i l amidase i n r i c e p l a n t s and i n r i c e root suspension c u l t u r e s has been i n v e s t i g a t e d (20, 45). The a c t i v i t y of the enzyme was found to be two to four times greater i n o l d e r p l a n t s (four leaves) than i n younger p l a n t s ( l e s s than four l e a v e s ) . P r o p a n i l amidase was a l s o demonstrated i n the r i c e suspension c u l t u r e , but i n t e r e s t i n g l y , the enzymatic a c t i v i t y could only be demonstrated a f t e r the t i s s u e c u l t u r e had developed to s t a t i o n a r y phase (5.5 days). This i n v e s t i g a t i o n i s important because i t documents a change i n the b i o s y n t h e t i c capacity of p l a n t t i s s u e c u l t u r e s w i t h the age of the c u l t u r e . The dependency of s e v e r a l enzymes, i n c l u d i n g phenylalanine ammonia l y a s e , upon i l l u m i n a t i o n of p a r s l e y c e l l c u l t u r e s a l s o has been shown (46). The metabolism of diphenamid (N-N-dimethyl-2,2-diphenylacetamide) by soybean r o o t t i p c e l l suspension c u l t u r e s has been

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

M U M M A

A N D H A M I L T O N

Plant Tissue and Cell

Cultures

45

i n v e s t i g a t e d (19) a t d i f f e r e n t stages o f growth and compared with whole p l a n t s . The metabolites found i n both p l a n t s and suspension c u l t u r e s were N-hydroxymethyl-N-methyl-2,2-diphenylacetamide (MODA), N-methyl-2,2-diphenylacetamide (MMDA), 2,2-diphenylacetamide (DA) and two p o l a r g l y c o s i d e s . One of the g l u c o s i d e s was a c i d i c and was i d e n t i f i e d as an e s t e r of malonic a c i d (Figure 2).

DA

Figure 2.

MMDA

(Acidic Glycoside)

Metabolism of diphenamid by soybean root cells in suspension culture

About 9-22% o f the diphenamid was metabolized by the c e l l c u l t u r e s at e a r l y l o g (3-7 days) and s t a t i o n a r y phases (14-18 days), r e s p e c t i v e l y . However, diphenamid metabolism per gram was about 2 times more r a p i d by e a r l y l o g phase c e l l s than by stationary c e l l s . Cultures o f a l l ages formed the same metabol i t e s with MODA and the d e a l k y l a t e d products predominating. The r e l a t i v e composition o f the metabolites i s presented i n Tables I I I and IV. The hydroxylated m e t a b o l i t e , MODA, was found almost e x c l u s i v e l y i n the medium 92-99%, and the d e a l k y l a t e d products were found predominantly i n the medium (68-94%). Log phase and s t a t i o n a r y phase c e l l s produced l a r g e r amounts of the d e a l k y l a t e d metabolites per mg dry weight per day than d i d e a r l y l o g phase c e l l s . G l y c o s i d e s c o n s i s t e d of only 6-7% of the t o t a l metabolites i n c e l l c u l t u r e s but were the major metabolites (46-48%) of tomato, pepper and soybean p l a n t s . These c e l l suspension c u l t u r e s demonstrate c l e a r l y the same metabolic degradation pathways as i n t a c t p l a n t s , but s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s do occur e s p e c i a l l y i n the small amount of g l y c o s i d e formation by the soybean root suspension c e l l s . No q u a l i t a t i v e changes occurred i n the metabolism of diphenamid with age of the c u l t u r e . The metabolism of c i s a n i l i d e ( c i s - 2 , 5 - d i m e t h y l - l - p y r r o l i d i n e c a r b o x a n i l i d e ) , a s e l e c t i v e preemergence h e r b i c i d e , has been i n v e s t i g a t e d i n excised leaves and c e l l suspension c u l t u r e s of

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

46

XENOBIOTIC

M E T A B O L I S M

Table I I I . Diphenamid Metabolism by Growth Phase of Soybean C e l l Suspension c u l t u r e s (19)

Growth Phase

Diphenamid or M e t a b o l i t e

nmol per Gram C e l l s Medium C e l l Extract

E a r l y Log (3-7 Days)

Diphenamid MODA Dealkylate Glucosid

12.8 0.1

444.8 31.6

Log (7-14 Days)

Diphenamid MODA Dealkylated Glucoside

41.9 2.2 10.6 1.9

99,4 27.4 27.8 1.1

Stationary (14-18 Days)

Diphenamid MODA Dealkylated Glucoside

42.5 1.2 6.7 0.9

144.3 25.4 14.5 1.9

Table IV. R e l a t i v e Composition of Diphenamid or Metabolites i n C e l l s and Medium i n E a r l y Log Phase (19) Diphenamid or M e t a b o l i t e

% C e l l Extract

Diphenamid MODA Dealkylated Glucoside A c i d i c Glucoside

2,.8 0..3 5.,5 25,,0 100..0

Medium 97.,2 99.,7 94,,5 75.,0 0.,0

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

M U M M A

A N D

Plant Tissue and Cell

H A M I L T O N

Cultures

47

c a r r o t and cotton (17, 18). A f t e r 6 days, excised c a r r o t leaves metabolized ca. 70% o f the a p p l i e d c i s a n i l i d e , but c a r r o t t i s s u e c u l t u r e s metabolized >95% o f the a p p l i e d h e r b i c i d e i n 3 days. Cotton leaves metabolized c i s a n i l i d e t o a greater extent than cotton suspension c u l t u r e s . The metabolism of c i s a n i l i d e by c a r rot and cotton p l a n t s i s shown i n Figure 3 and the r e l a t i v e comp o s i t i o n of metabolites i n p l a n t s and t i s s u e c u l t u r e i s presented i n Table V.

cri

CH

3

Glycoside I

3

Glycoside H Pesticide Biochemistry and Physiology

Figure 3.

Metabolism of cisanilide by carrot and cotton phnts and cells in culture

In the excised p l a n t s , g l y c o s i d e s I and I I were the major methanol s o l u b l e metabolites and only t r a c e q u a n t i t i e s of the aglycons were detected. In c a r r o t and cotton c e l l suspension c u l t u r e s , aglycon I or g l u c o s i d e I was not detected and only t r a c e amounts of g l y c o s i d e I I were detected. Aglycon I I was a major metabolite i n the medium of the c e l l suspension c u l t u r e s . In the excised p l a n t s , l e s s (20-25%) of the * C - l a b e l was found i n the i n s o l u b l e residue f r a c t i o n compared to the c e l l suspension c u l t u r e s (39-40%). ll

American Chemical Society Library

1155 16th St. N. W. In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; Washington, D. C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

V. a

a

Methanol Soluble 25 13 (Unknown) I n s o l u b l e Residue 18 39 G l y c o s i d e I was not detected and g l y c o s i d e I I was t e n t a t i v e l y as a minor component.

identified

Excised Cotton Leaves (2 Days) Cotton Suspension Cultures (7 Days) Fraction % % Cells % Medium T o t a l Metabolized >95 >76 Methanol Soluble 55 — 24 (Glycosides I>II) (Aglycon I I )

40

Carrot Suspension C u l t u r e (3 D a y s ) % Cells % Medium >95 — 40 (Aglycon I I ) 20

R e l a t i v e Composition of C i s a n i l i d e M e t a b o l i t e s (18).

Excised Carrot Leaves (6 Days) Fraction % T o t a l Metabolized 70 Methanol Soluble 50 (Glycoside 1=11) Methanol Soluble 10 (Unknown) I n s o l u b l e Residue 20

Table

2.

M U M M A

A N D H A M I L T O N

Plant Tissue and

Cell

Cultures

49

At f i r s t approximation, i t would be easy to conclude that the metabolic degradation pathways were d i f f e r e n t i n the excised p l a n t s versus the c e l l suspensions s i n c e aglycon I and g l u c o s i d e I were not detected i n the t i s s u e or the medium. T h i s d i f f e r e n c e may i n d i c a t e r a p i d i n c o r p o r a t i o n of aglycon I i n t o a methanoli n s o l u b l e f r a c t i o n , because when aglycon I was administered to the c e l l suspension c u l t u r e , i t was r a p i d l y converted to methanol i n s o l u b l e products. Neither t i s s u e i s able to cleave a p p r e c i a b l y the urea type s t r u c t u r e of c i s a n i l i d e . T h i s i s a l s o the case f o r the metabolism of other urea h e r b i c i d e s by p l a n t s (43). Both t i s s u e s possess the a b i l i t y to form the hydroxy 1 d e r i v a t i v e , aglycon I I , but the c e l l c u l t u r e s e v i d e n t l y have a reduced c a p a c i t y to form the g l y c o s i d e conjugate and consequently aglycon I I accumulate metabolite i s apparentl s o l u b l e p r o d u c t ( s ) . Thus, the metabolic pathways may be somewhat s i m i l a r except f o r l o s s of aglycons i n t o the medium and the increased formation of an i n s o l u b l e product. The p h y t o t o x i c i t y and metabolism of the h e r b i c i d e m e t r i b u z i n (4-amino-6-t-butyl 3-[methylthio]-as-triazin-5-[4H]-one) has been i n v e s t i g a t e d w i t h dark-grown soybean cotyledon suspension c u l t u r e s from s u s c e p t i b l e ("Coker 102") and r e s i s t a n c e ("Bragg") c u l t i v a r s . Bioassays were based on p o p u l a t i o n changes of v i a b l e c e l l s during i n c u b a t i o n with m e t r i b u z i n . V i a b l e c e l l s were c l a s s i f i e d as c e l l s with s t r u c t u r a l i n t e g r i t y and cytoplasmic streaming. Differential r e s i s t a n c e to m e t r i b u z i n was demonstrated by the c e l l suspensions from r e s i s t a n t and s u s c e p t i b l e c u l t i v a r s . M e t r i b u z i n had been reported to i n h i b i t photosynthesis, but the demonstrated phytot o x i c i t y toward both c u l t i v a r s of dark-grown a c h l o r o p h y l l o u s suspension c u l t u r e s i n d i c a t e d that p h y t o t o x i c i t y was not r e s t r i c t e d to photosynthesis. D e t o x i f i c a t i o n of m e t r i b u z i n by soybeans has been a t t r i b u t e d to formation of an N-glucoside. Enzymatic detoxi f i c a t i o n of m e t r i b u z i n d i d not occur i n the s u s c e p t i b l e c u l t i v a r due to the accumulation of a substance which i n h i b i t e d the enzyme. The r e s i s t a n t c u l t i v a r metabolized the i n h i b i t o r to a n o n i n h i b i tory form. Therefore, m e t r i b u z i n r e s i s t a n c e by the Bragg c u l t i v a r was a t t r i b u t e d to the a b i l i t y of t h i s c u l t i v a r to metabolize a common enzymatic i n h i b i t o r . P l a n t t i s s u e c u l t u r e techniques have been used by numerous i n v e s t i g a t o r s (23 - 29) f o r 2,4-D metabolism s t u d i e s . In 1968, the metabolism of 2,4-D-2-i^C by suspension c u l t u r e s of soybean root grown under continuous l i g h t (2000 lux) was examined (23). Most of the C - l a b e l i n the t i s s u e appeared as two spots on paper chromatography. The f a s t e r moving compound has the same Rf as 2,4-D and was assumed to be f r e e 2,4-D. The slower moving spot was a g l y c o s i d e that y i e l d e d glucose and f r e e 2,4-D when t r e a t e d w i t h emulsin. These i n v e s t i g a t o r s assumed that 2,4-D was metabol i z e d only to the 3-D glucose e s t e r of 2,4-D. Presumably, the amino a c i d conjugates, which have been subsequently i d e n t i f i e d as metabolites of 2,4-D, d i d not separate from 2,4-D i n the 1 4

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

50

XENOBIOTIC

M E T A B O L I S M

chromatographic s o l v e n t s used. The a d d i t i o n o f glutamine to the media increased the uptake of 2,4-D, but d i d not change the apparent metabolic products when analyzed by t h e i r chromatographic s o l vent systems. Some 2,4-D was a l s o a s s o c i a t e d with p r o t e i n . Two clones of f i e l d bindweed (Convolvulus a r r e n s i s L.) that d i f f e r e d i n t h e i r s u s c e p t i b i l i t y to 2,4-D under f i e l d and greenhouse c o n d i t i o n s a l s o e x h i b i t e d s i m i l a r d i f f e r e n c e s when stem c e l l s were c u l t u r e d i n l i q u i d and agar media (30). When amino a c i d s were added to the c u l t u r e media, the response to 2,4-D was a l t e r e d . The a b s o r p t i o n o f 2,4-D was increased w i t h glutamine and decreased with glutamic a c i d . Glutamic a c i d increased the t o l e r ance of the s u s c e p t i b l e clone, but reduced the t o l e r a n c e of the r e s i s t a n t clone. Glutamine i n c r e a s e d the s u s c e p t i b i l i t y of the s u s c e p t i b l e clone to a much g r e a t e r degree than i t d i d the r e s i s tant clone. There was and n i t r a t e reductase a c t i v i t y When soybean (Glycine max L.) cotyledon c a l l u s was incubated for 32 days w i t h 2,4-D-l- ^C,metabolites changed q u a l i t a t i v e l y and q u a n t a t i v e l y w i t h time (24). The water s o l u b l e f r a c t i o n from the t i s s u e increased i n r a d i o a c t i v i t y . When i t was t r e a t e d with 3 glucosidase, at l e a s t e i g h t aglycons that changed with time were r e l e a s e d ( F i g . 4 ) . 4-Hydroxy-2,5-dichlorophenoxyacetic a c i d (4OH-2,5-D) was the most abundant aglycon and 4-hydroxy-2,3-dichlorophenoxyacetic a c i d (4-OH-2,3-D) was i d e n t i f i e d as a minor component. Free 2,4-D a l s o was l i b e r a t e d f o l l o w i n g enzymatic treatment. The presumed presence o f 2,4-dichlorophenoxyacetyl-3-0-D glucose, a metabolite reported p r e v i o u s l y (23), was suggested. A

The ether s o l u b l e f r a c t i o n reached a maximum a f t e r 2 days and c o n s i s t e d of seven d i f f e r e n t regions of components on paper chromatography ( E t i - E t 7 ) that v a r i e d with time ( F i g . 5 ) . The major component (Eti*) was i d e n t i f i e d as the glutamic a c i d conjugate of 2,4-D and i t s r e l a t i v e composition was maximal a f t e r one day. The a s p a r t i c a c i d conjugate o f 2,4-D ( E t 2 ) increased g r a d u a l l y i n r e l a t i v e composition. S u r p r i s i n g l y , f r e e 2,4-D ( E t 7 ) d i d not reach i t s maximum u n t i l eight days. These data imply that contrary to some previous s t u d i e s , the metabolism of 2,4-D by plant t i s s u e i s q u i t e complex. Subsequently, f i v e a d d i t i o n a l amino a c i d conjugates of 2,4-D have been i d e n t i f i e d from soybean (27). These i n clude the a l a n i n e , v a l i n e , l e u c i n e , phenylalanine and tryptophan conjugates. A t y p i c a l d i s t r i b u t i o n of 2,4-D metabolites i s o l a t e d from 4-week-old c a l l u s t i s s u e i s presented i n Table VI.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

M U M M A

Plant Tissue and Cell

A N D H A M I L T O N

Cultures

ΙΟΟ r

80

u. 60 I UJ

i

$ 40 I

2CLU

4

8

16

INCUBATION TIME (DAYS) Plant Physiology Figure 4. The relative amounts of the aglycons obtained from the water-soluble fractions of soybean callus tissue incubated with 2,4-D-l- C: (Agi), primarily 4-OH-2,5-D and 4-011-2,3-D; (Ag ), 2,4-D. 14

7

Table V I .

R e l a t i v e Percentage of 2,4-D M e t a b o l i t e s i n Soybean C a l l u s T i s s u e (27). a

Ether-Soluble Metabolite Unk

Metabolites % In T i s s u e 1.2

2,4-D-Asp 2,4-D-Gly Unk 2,4-D-Ala,-Val) 2,4-D 2,4-D-Leu,-Phe -Try) TOTAL a

3.7 12.9 1.4 5.3 33.7 4.0 62.2%

Four-week-old c a l l u s t i s s u e with 2,4-D.

(emulsin) Aglycons % In Tissue Metabolite 26.3 (4-OH-2,5-D, 4-OH-2,3-D) 2.5 Unk 1.1 Unk 1.0 Unk 0.8 Unk 0.9 Unk 0.8 2,4-D 0.4 Unk 33.8% (10g) incubated

f o r 8 days

Journal of Agricultural and Food Chemistry

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

52

XENOBIOTIC

I

2

4

8

INCUBATION TIME

16

24

M E T A B O L I S M

32

(DAYS)

Plant Physiology Figure 5. Relative amounts of ether solubles isolated from soybean callus tissues incubated with 2,4-D-l- C: (Et ), aspartic acid conjugate; (Et ), glutamic acid conjugate; and (Et ), free 2,4-D. 14

2

k

7

A d d i t i o n a l minor aglycons a l s o have been i d e n t i f i e d t e n t a t i v e l y from corn endosperm c a l l u s as 3-hydroxy-2,4-dichlorophenoxyacetic a c i d (3-OH-2,4-D), 4-hydroxy-2-chlorophenoxyacetic a c i d (4-OH-2-C1) (27), and from wheat suspension c u l t u r e s as 6-hydroxy2,4-dichlorophenoxyacetic a c i d (6-OH-2,4-D) and 2-hydroxy-4chlorophenoxyacetic a c i d (2-OH-4-C1) (29). The e t h y l ester o f 2,4-D has been i s o l a t e d from the g l y c o s i d e f r a c t i o n of r i c e c a l l u s t i s s u e c u l t u r e f o l l o w i n g (3-glucosidase treatment (28). The e t h y l ester was presumed to be an a r t i f a c t of the i s o l a t i o n procedure probably being derived from the glucose e s t e r that e x i s t s i n high concentration i n t h i s t i s s u e . The glutamic a c i d conjugate of 2,4-D i s not an end product of 2,4-D metabolism (25) . When C-2,4-D-glutamic a c i d was i n c u bated with soybean c a l l u s t i s s u e , f r e e 2,4-D, the a s p a r t i c a c i d conjugate, and other products were found. These data suggest that amino a c i d conjugates may represent a r e s e r v o i r of bound 2,4-D 1l +

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

M U M M A AND HAMILTON

Plant Tissue and Cell

Cultures

53

that may be i n v o l v e d i n the r e g u l a t i o n of 2,4-D l e v e l s i n the tissue. Comparative metabolism s t u d i e s o f 2,4-D i n c a r r o t , jackbean, sunflower, tobacco, corn and r i c e c a l l u s t i s s u e c u l t u r e s (27, 28) and a wheat suspension c u l t u r e (29) a r e shown i n Tables V I I and V I I I . A l l t i s s u e s examined, except r i c e , formed amino a c i d conjugates and a l l formed g l y c o s i d e s (phenolic g l y c o s i d e s as w e l l as glucose e s t e r s ) . The d i c o t s formed a higher r e l a t i v e percentage of amino a c i d conjugates w h i l e the monocots produced a higher percentage o f g l y c o s i d e s . The metabolism o f 2,4-D i n soybean and corn p l a n t s has been compared w i t h 2,4-D metabolism i n soybean c a l l u s t i s s u e (26). These data are presented i n Tables IX and X. In t h i s experiment, the 2,4-D-l- *C was d i r e c t l injected int th c a l l u tissu growing on agar r a t h e r tha ed c a l l u s . The c a l l u s growth and i t s n u t r i e n t s t a t u s was not changed. The metabolites found i n the p l a n t s a r e a l s o present i n the c a l l u s t i s s u e , but differ quantitatively. Of p a r t i c u l a r note i s the f a c t that f r e e hydroxylated 2,4-D e x i s t s i n both c a l l u s and i n p l a n t s i n c o n t r a s t to e a r l i e r t i s s u e c u l t u r e experiments. Free hydroxylated 2,4-D a l s o has been reported i n bean p l a n t s (48). These experiments a l s o suggest that the unknown compounds, Unki and U n k 2 , a r e amino a c i d conjugates o f hydroxylated 2,4-D metabolites ( p r i m a r i l y the glutamic a c i d conjugate of 4-OH-2,5-D) and are common to a l l t i s sue examined. A summary of the metabolism of 2,4-D i n p l a n t s i s presented i n F i g u r e 6. Recently, the metabolism of 2,4-D has been reported i n s i x i n t a c t p l a n t s : wheat, timothy, green bean, soybean, sunflower and strawberry (47). The r e l a t i v e percentage of amino a c i d conjugates i s low compared to the p r e v i o u s l y c i t e d work w i t h t i s s u e c u l t u r e experiments (Table X I ) . Of p a r t i c u l a r n o t i c e i s the unusually h i g h percentage of the d i c h l o r o p h e n o l g l y c o s i d e i n strawberry. In c o n t r a s t to use of t r u e suspension c u l t u r e s by most other l a b o r a t o r i e s , Feung e t a l . (24-28) u s u a l l y incubated a f a i r l y l a r g e amount (~10 gms) of small p i e c e s of 4-5 week o l d c a l l u s taken from s o l i d medium i n 25-40 ml l i q u i d medium w i t h shaking during treatment w i t h 2,4-D-l- ^C. T h i s technique r e s u l t s i n n e a r l y t o t a l uptake o f the a p p l i e d 2,4-D w i t h i n 48 hours, the accumulation o f s i g n i f i c a n t amounts of g l y c o s i d e metabolites i n the t i s s u e (phenolic g l y c o s i d e s and glucose e s t e r ) , i n s i g n i f i c a n t amounts of primary hydroxylated products and no accumulation of products i n the medium. In c e l l suspension c u l t u r e s , a b s o r p t i o n a l s o i s r a p i d but metabolites o f t e n accumulate i n the medium. In the study (26) where c a l l u s t i s s u e was i n j e c t e d d i r e c t l y w i t h 2,4-D-l- C., a d d i t i o n a l metabolites were found. Thus, the method of p e s t i c i d e a d m i n i s t r a t i o n may be important. Data w i t h i n t a c t p l a n t s (Tables IX, X and XI) a r e c o n s i s t a n t w i t h metabolism data obtained w i t h p l a n t t i s s u e c u l t u r e s , but s i g n i f i c a n t q u a n t i t a t i v e d i f f e r e n c e s do occur. The monocots ll

1

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

ll+

a

4.1 0.8 0.5 0.8 0.6 3.2

2.6 0.4 0.3 0.3 1.1 1.2

12.8

3.2 0.7 0.4 0.1

0.9 1.0

13.2

42.2

2.6 7.7

8.3 2.0 1.9 2.3

17.4

64.6

10.2

1.6

19.4 0.5 1.2

31.7

Corn

A l l were c a l l u s t i s s u e c u l t u r e s (27, 28) except wheat (29).

18.3

8.3

6.9

6.9

(4-OH-2,3-D, 4-OH-2,5-D) Unk Unk Unk Unk Unk Unk 2,4-D Ethyl-2,4-D Others TOTAL

% Total i n Tissue Sunflower Tobacco

Carrot

Jackbean

14.9 12.9 1.5 29.7

0.4

Rice

23.9

Ν/

10. 2

\

Wheat

R e l a t i v e Percentage of Water-Soluble 2,4-D-l- C M e t a b o l i t e s I s o l a t e d from Seven Species of Plant T i s s u e C u l t u r e s as the Aglycons.

Metabolites

Table V I I .

M U M M A AND H A M I L T O N

ω

Φ

4J cd φ

φ

ο

ω cd

S r-l

ο ο

Φ β

ω • β CO φ

• m rH

00

co m

00 m vO rH

m

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co

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M

> Φ

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cd H

Φ

43 4J

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Φ

φ

CO

r>.

Ο

m Ο

Μ-Ι β β

/—Ν

•

UO rH m m rH

00

οο

Ι

CM| «\

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β

ω φ CM

m rH CM ->

> U ο 4J cd υ

•Η

Φ

CM CM rH

Ο

cd φ rÛ

G Β u ο Φ u Ρ-ι -triazines. The i s o l a t e d GSH jS-transferase from pea was s t a b l e , but crude enzyme preparations from corn, peanut and c o t t o n underwent i r r e v e r s i b l e i n h i b i t i o n when s t o r e d f o r s e v e r a l hours a t 4 ° . Enzyme a c t i v i t y was detected i n higher concentrations i n r e s i s t a n t species ( c o t t o n , corn, peanut, pea, soybean and okra) than i n s u s c e p t i b l e s p e c i e s (cucumber, tomato and squash). F l u o r o d i f e n s e l e c t i v i t y appeared to be based on enzyme d i s t r i b ­ u t i o n and c o n c e n t r a t i o n . A broad s u b s t r a t e s p e c i f i c i t y study was not conducted w i t h t h i s enzyme, but two other diphenylether h e r b i c i d e s were t e s t e d and found to be i n a c t i v e . I t was hypo­ t h e s i z e d that only diphenylethers that were h i g h l y a c t i v a t e d a t the C - l p o s i t i o n would a c t as s u b s t r a t e s . Mercaptoethanol, 2,3-dimercaptopropanol, d i t h i o t h r e i t o l and c y s t e i n e would not f u n c t i o n as the s u l f h d r y l s u b s t r a t e with the enzyme from pea. Recent s t u d i e s have shown that the f u n g i c i d e PCNB (pentachloronitrobenzene) i s a l s o converted to GSH conjugates w i t h an enzyme system i s o l a t e d from pea (145). A d d i t i o n a l s t u d i e s w i l l be needed to determine i f the same enzyme i s i n v o l v e d i n the metabolism of both PCNB and f l u o r o d i f e n . A unique f e a t u r e of the

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC M E T A B O L I S M

108

PCNB GSH S - t r a n s f e r a s e assay system was the i n c l u s i o n of t e r t ^ butanol to i n c r e a s e PCNB s o l u b i l i t y and enzymatic a c t i v i t y (Figure 14). The use of t e r t - b u t a n o l i n enzyme systems has been p r e v i o u s l y reported (151). T h i s system has not been completely s t u d i e d w i t h r e s p e c t to the PCNB-GSH conjugation r e a c t i o n . D e t a i l e d i n h i b i t o r s t u d i e s were conducted w i t h the GSH S~ t r a n s f e r a s e s i s o l a t e d from both corn and pea (Table X ) . Both enzymes gave s i m i l a r responses to s u l f h y d r y l compounds and to the c l a s s i c a l i n h i b i t o r s of mammalian GSH S - t r a n s f e r a s e a c t i v i t y , sulfobromophthalein and 1,2-dichloronitrobenzene. I n h i b i t i o n by sulfobromophthalein was competitive i n mammalian systems and a l s o appeared to be competitive i n both p l a n t systems, Propachlor ( 2 - c h l o r o - N - i s o p r o p y l a c e t a n i l i d e ) and barban (4-chloro~2-butynyl*m-chlorocarbanilate) were i n h i b i t o r s of the enzymatic r e a c t i o n s with a t r a z i n e and f l u o r o d i f e n was a l s o observed. The competitive i n h i b i t o r of both enzymes suggested some commonality of the a c t i v e s i t e ( s ) . On the other hand, the f a c t that a t r a z i n e was n e i t h e r a s u b s t r a t e nor an i n h i b i t o r of the pea enzyme suggested important d i f f e r e n c e s . I n h i b i t i o n of the pea enzyme by other diphenylether compounds, phenylureas and acetamide h e r b i c i d e s suggested the p o s s i b i l i t y of p e s t i c i d e i n t e r a c t i o n s w i t h these compounds. A number of s - t r i a z i n e s were t e s t e d as i n h i b i t o r s of the corn enzyme. These s t u d i e s suggested that the bis(alkylamino)-methoxy-s-1riaz ines and the methylmercapto analogs were probably competitive i n h i b i t o r s capable of b i n d i n g at the a c t i v e s i t e , but i n c a p a b l e of undergoing r e a c t i o n , Hydrox y t r i a z i n e s and the d e a l k y l a t e d t r i a z i n e s were not e f f e c t i v e inhibitors. I t i s of p a r t i c u l a r i n t e r e s t to note that a known s y n e r g i s t o f a t r a z i n e , 2 , 3 , 6 - t r i c h l o r o p h e n y l a c e t i c a c i d , was an i n h i b i t o r of the GSH S - t r a n s f e r a s e from corn. EPTC ( S - e t h y l dipropylthiocarbamate) does not appear to be a GSH S - t r a n s f e r a s e s u b s t r a t e . However, a f t e r o x i d a t i o n to the s u l f o x i d e , i t r e a d i l y undergoes conjugation i n the presence of GSH S - t r a n s f e r a s e s i s o l a t e d from r a t l i v e r (152) or corn r o o t (144). 0 tt

0 0 f C2H5-S-C-NCC3H7)2 M

C H5-S-C-N(C3H )2 2

7

EPTC

EPTC S u l f o x i d e

0 It GSH-transferase EPTC S u l f o x i d e

;-cS-Carbamyl^-GSH

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3.

LAMOUREUX A N D FREAR

Plant Enzyme

109

Studies

14 -,

12 -I

ίο 1

φ ε

6 J

4 J

2Η

ImM GSH 9.2 μΜ PCNB 120 mM Pi 0.32 mg enzyme

TO

Τ 15

—ι 20

% (v/v) tert-butanol in system Figure 14.

Effect of tert-butanol on GSH conjugation of PCNB catalyzed by an enzyme system isolated from pea (192)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 72 0

29 29 34 41 54 —

86 0 3 —

—

28 60 88 79 55 57 68

1 1 0.1 0.06

0.1 0.1 0.1 0.1 1 0.03 0,1 0.1 1.0

2,3-dimercaptopropanol

cysteine

atrazine

2,4-bis(isopropylamino-6-methylmercapto-s-triazine*

2,3,6-trichlorophenyl acetic acid

propachlor

barban

sulfobromophthalein

1,2-dichloro-4-nitrobenzene

nitrofen*

diuron*

propanil

l-chloro-3-tosylamldo-7-amino-L-2 2-heptane«HCl* ( a l k y l a t i n g agents)

*Related compounds were t e s t e d w i t h s i m i l a r r e s u l t s .

58

11

6

1

S-methyl g l u t a t h i o n e

61

N.A.

40

14

1

dithiothreitol

Inhibitor

% I n h i b i t i o n of GSH-transferase (corn)

% I n h i b i t i o n of GSH-transferase (pea)

I n h i b i t o r s t u d i e s w i t h the g l u t a t h i o n e S-transferases i s o l a t e d from corn (143) and pea (142). Inhibitor concentration (mM)

Table X.

3.

L A M O U R E U X AND FREAR

Phnt

Enzyme

Studies

111

T h i s i s comparable to a previous observation that a methylm e r c a p t o - s - t r i a z i n e would not undergo conjugation u n t i l a f t e r o x i d a t i o n t o the s u l f o x i d e (149). Recent s t u d i e s have a l s o shown that chlorpropham ( i s o p r o p y l m-chlorocarbanilate) i s not a s u b s t r a t e f o r GSH S-transferase a c t i v i t y i n oat, but i t s o x i d a t i o n product, 4-hydroxychlorpropham, r e a d i l y undergoes an enzymatic r e a c t i o n w i t h c y s t e i n e or GSH (139, 140). These observations i l l u s t r a t e the need to consider the p o s s i b l e r o l e of a c t i v a t i n g r e a c t i o n s i n GSH conjugation. When corn i s t r e a t e d w i t h Ν,N-diallyl-2,2-dichloroacetamide (R-25788), h e r b i c i d a l i n j u r y due to EPTC i s g r e a t l y reduced (153). Glutathione J3-transferase a c t i v i t y and the c o n c e n t r a t i o n o f GSH were increased 2- to 3 - f o l d by treatment w i t h 0.3 to 30 ppm of R-25788. I t was concluded that decreased h e r b i c i d a l i n j u r y was due to an increased r a t elevated l e v e l s of GSH EPTC-susceptible o a t s e e d l i n g s , the l e v e l s o f GSH and GSH j>t r a n s f e r a s e d i d not i n c r e a s e i n response t o R-25788. The a c t i o n of R-25788 appears t o be s e l e c t i v e . No i n c r e a s e i n a c t i v i t y was noted when the i s o l a t e d corn system was t r e a t e d w i t h R-25788; t h e r e f o r e , R-25788 does not appear to be a simple a c t i v a t o r . Increased l e v e l s o f GSH ^ - t r a n s f e r a s e a c t i v i t y were observed i n both crude and p a r t i a l l y p u r i f i e d enzyme preparations a f t e r treatment w i t h R-25788. Although i t was not proven, the r e s u l t s suggest that enzyme i n d u c t i o n o r p o s s i b l e removal o f endogenous i n h i b i t o r s may be r e s p o n s i b l e f o r the observed increases i n enzyme a c t i v i t y . Twenty-eight compounds were compared to R-25788 f o r t h e i r e f f e c t i v e n e s s i n i n c r e a s i n g GSH S-transferase a c t i v i t y and GSH content i n corn s e e d l i n g r o o t s . Although s i g n i f i c a n t exceptions were noted, the e f f e c t i v e n e s s of these compounds as a n t i d o t e s g e n e r a l l y c o r r e l a t e d w i t h increased GSH and GSH Sr-transferase l e v e l s . Chlorpropham ( i s o p r o p y l m-chlorocarbanilate) and c i s a n i l i d e ( c i s - 2 , N - p h e n y l - l - p y r r o l i d i n e c a r b o x a n i l i d e ) a r e metabolized to hydroxylated d e r i v a t i v e s i n c e r t a i n p l a n t species (139, 154). Recent evidence i n d i c a t e s that the 4-hydroxylated d e r i v a t i v e s of chlorpropham and c i s a n i l i d e a r e converted to GSH and c y s t e i n e conjugates i n oat shoot s e c t i o n s (139, 140) (Figure 15). The s o l u b l e enzyme complex that c a t a l y z e s conjugate formation was i s o l a t e d from oat. When c y s t e i n e and 4-hydroxychlorpropham were incubated with the enzyme, a p o l a r metabolite was formed. When GSH was s u b s t i t u t e d f o r c y s t e i n e , a more p o l a r product was formed. The i n v i t r o enzyme system was used to produce s u f f i c i e n t metabolite from the r e a c t i o n w i t h c y s t e i n e and 4-hydroxychlor­ propham to allow i s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of the product (139). Because o f the low y i e l d o f t h i s product i n the i n v i v o system and d i f f i c u l t i e s encountered i n i t s i s o l a t i o n , the use of an i n v i t r o system f o r product formation g r e a t l y f a c i l i t a t e d the c h a r a c t e r i z a t i o n of t h i s product. In the c h a r a c t e r i z a t i o n o f the c y s t e i n e conjugate, c y s t e i n e C-S_ l y a s e

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC

112

METABOLISM

SG

Figure 15.

Reactions thought to he catalyzed by a GSH/cysteine from oat (139,140)

S-transferase

R-S-CH -C-C00H 2

NH

2

-[c-s (ORGANIC)

LYASE]

(H 0) 2

0 0

RSH CH

II II -C-C-OH ^ - N A D H

MASS

f [LACTIC \~~[ DEHYDROGENASE |

SPECTROMETER

^^NAD OH I CH,-C-COOH I H 3

Δ Α 340nm Figure 16.

Cysteine C-S lyase cleavage of cysteine conjugates (139,150)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3.

L A M O U R E U X AND FREAR

Plant Enzyme

Studies

113

was used to cleave the c y s t e i n e moiety from the a r y l group to y i e l d p y r u v i c a c i d and a thiophenol (Figure 16). The r e a c t i o n was q u a n t i t a t i v e and the l i b e r a t e d p y r u v i c a c i d was measured by c o u p l i n g the c y s t e i n e £-S l y a s e r e a c t i o n to a l a c t i c a c i d dehydrogenase r e a c t i o n . These coupled r e a c t i o n s were p r e v i o u s l y used i n the c h a r a c t e r i z a t i o n of a GSH-related conjugate of a t r a z i n e (150). Glutathione jS-transferase a c t i v i t y w i t h GSH and 4-hydroxychlorpropham was not demonstrated u n t i l the crude enzyme was p a r t i a l l y p u r i f i e d by Sephadex g e l chromatography. This behavior suggested the presence of endogenous i n h i b i t o r s . D e t a i l e d i n h i b i t o r s t u d i e s showed that s e v e r a l n a t u r a l l y o c c u r r i n g aromatic compounds and 3-chloro-4-hydroxyaniline were powerful i n h i b i t o r s of th c y s t e i n S-transferas activit (155) I t was suggested that thes o c c u r r i n g hydroxylated aromati x e n o b i o t i c s as s u b s t r a t e s . Two t r a n s f e r a s e enzyme systems were apparently present i n oat shoots. One e x h i b i t e d n e a r l y comparable a c t i v i t y with e i t h e r c y s t e i n e or GSH and the other d i s p l a y e d much greater a c t i v i t y w i t h c y s t e i n e . The l a t t e r enzyme a l s o functioned as a GSH S-transferase when the e t h y l e s t e r of c y s t e i n e was added to the r e a c t i o n mixture (140). The nature and the s i g n i f i c a n c e of t h i s a c t i v a t i o n i s not understood. These are the f i r s t s t u d i e s to suggest that c y s t e i n e may be u t i l i z e d as a s u b s t r a t e much l i k e GSH i n a t r a n s f e r a s e r e a c t i o n (139, 140, 155). T h i s system should be studied i n greater d e t a i l to b e t t e r evaluate i t s s i g n i f i c a n c e to x e n o b i o t i c metabolism. Glutathione j>-transferase a c t i v i t y was r e c e n t l y i s o l a t e d from 10 a g r i c u l t u r a l l y important p l a n t species and screened f o r a c t i v i t y with 8 d i f f e r e n t p e s t i c i d e substrates (146). Of the substrates examined, GSH S-transferase a c t i v i t y was demonstrated i n a l l species w i t h PCNB, propachlor and CDAA ( N , N - d i a l l y l chloroacetamide). The r e s u l t s suggested that c e r t a i n types of GSH j>-transferase a c t i v i t y may be widely d i s t r i b u t e d i n higher plants. These l i m i t e d s t u d i e s have c l e a r l y shown that GSH S-transf e r a s e s p l a y an important r o l e i n x e n o b i o t i c metabolism i n p l a n t s . Some GSH S-transferases appear to be widely d i s t r i b u t e d i n the p l a n t kingdom, but others appear to be more l i m i t e d i n t h e i r distribution. Glutathione ^ - t r a n s f e r a s e enzymes p l a y an important r o l e i n the s e l e c t i v i t y of c e r t a i n h e r b i c i d e s , such as the 2 - c h l o r o - s - t r i a z i n e s , f l u o r o d i f e n and EPTC s u l f o x i d e , but t h e i r r o l e i n the s e l e c t i v i t y of h e r b i c i d e s such as the a - c h l o r o acetamides i s u n c e r t a i n . The p o s s i b i l i t y that h e r b i c i d a l s e l e c t i v i t y may be increased by s e l e c t i v i t y s t i m u l a t i n g or inducing GSH ^ - t r a n s f e r a s e l e v e l s has been r a i s e d . A d d i t i o n a l s t u d i e s are needed to determine the d i s t r i b u t i o n of GSH St r a n s f e r a s e s i n higher p l a n t s and to b e t t e r determine the p r o p e r t i e s of the i n d i v i d u a l t r a n s f e r a s e s .

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC

114 Glucose

METABOLISM

Conjugation:

The r e p o r t s that ethylenechlorohydrin was converted to a 3-0-D-glucoside and a g e n t i o b i o s i d e i n wheat (156) and tomato (157T were among the f i r s t i n d i c a t i o n s that p l a n t s had the a b i l i t y to convert c e r t a i n x e n o b i o t i c s to g l u c o s i d e s . I t was l a t e r shown that most higher p l a n t s (158, 159) had the a b i l i t y to convert exogenous phenols to $-j)-D-glueosides. T h i s a b i l i t y was apparently l a c k i n g i n algae, fungT and c e r t a i n aquatic p l a n t s (158). The formation of 0-, N-, and ^ - g l u c o s i d e s , a c y l a t e d g l u c o s i d e s , g e n t i o b i o s i d e s , and glucose e s t e r s have a l l been demonstrated e i t h e r i n v i t r o or i n v i v o with x e n o b i o t i c s or n a t u r a l substrates (160). The formation of glucosides i s extremely important i n p e s t i c i d e biochemistry f o r the f o l l o w i n g reasons: there i s a wid conjugation, g l u c o s i d e formatio terminal r e s i d u e , and g l u c o s y l a t i o n may play a r o l e i n p e s t i c i d e s e l e c t i v i t y or d e t o x i c a t i o n . The most common types of g l y c o s i d e r e a c t i o n s encountered i n p e s t i c i d e metabolism appear to i n v o l v e an i n i t i a l UDPG-dependent g l u c o s y l t r a n s f e r r e a c t i o n (Figure 17), The formation of simple 0-glucosides from polyhydroxylated phenols was demonstrated w i t h an i n v i t r o system from wheat germ (161). Substrate s p e c i f i c i t y t e s t s showed that the wheat germ g l u c o s y l t r a n s f e r a s e could use a number of polyhydroxy phenols as s u b s t r a t e s , but was not a c t i v e with simple phenols. A f t e r p u r i f i c a t i o n of t h i s enzyme, a c t i v i t y f o r c e r t a i n substrates was l o s t ; thus, the presence of more than one t r a n s f e r a s e was i n d i c a t e d . The i n v i t r o synthesis of 14 phenolic glucosides by crude enzymes from wheat germ and bean was compared with the i n v i v o s y n t h e s i s i n bean (162). The only products detected i n v i t r o were g e n e r a l l y the primary products formed i n v i v o . The enzyme systems from wheat germ and bean could not u t i l i z e simple mono-hydroxylated phenols as substrates; i t i s , t h e r e f o r e , questionable whether these enzymes are i n v o l v e d i n the formation of 3-0-D-glucosides from p e s t i c i d e s or p e s t i c i d e m e t a b o l i t e s . A number of UDPG:sterol g l u c o s y l t r a n s f e r a s e s have been i s o l a t e d from v a r i o u s p l a n t sources (163-166). These enzymes are u s u a l l y a s s o c i a t e d with the p a r t i c u l a t e f r a c t i o n (164). For some phenolic x e n o b i o t i c s , the p o s s i b i l i t y should be considered that UDPG:glucosyltransferase a c t i v i t y may be membrane bound. A UDPG-dependent enzyme that c a t a l y z e s the formation of β-0-D-glucosides with a v a r i e t y of phenols, a l k y l a l c o h o l s and other substrates has been i s o l a t e d from germinating mung bean (167). Attempts to demonstrate the presence of t h i s enzyme i n seedlings were not s u c c e s s f u l . The ammonium s u l f a t e f r a c t i o n a t e d enzyme from germinating mung beans could be stored i n l i q u i d n i t r o g e n with l i t t l e l o s s i n a c t i v i t y , but the more h i g h l y p u r i f i e d enzyme l o s t a l l a c t i v i t y upon f r e e z i n g . T h i s enzyme u t i l i z e d UDPG as the g l u c o s y l donor, had an estimated M.W. of 62,000 and had a pH optimum of approximately 10, The pH optimum

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3.

LAMOUREUX A N D FREAR

Plant Enzyme

115

Studies

3) 1-0 GLUCOSE ESTERS

4) Ν-GLUCOSIDES ,H

2

1

NADPH

F,HF H* 2

NADP

Figure 8. Schematic proposal for the various states of reduction of the flavo­ protein, NADPH-Cytochrome P-450 reductase. F, is presumed to represent FAD and F to represent FMN. The existence of various free radical species is indi­ cated. 2

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5.

ESTABROOK E T A L .

Type

I Metabolism

159

Table I

Concentration o f Microsomal Electron Transfer Components FMN P-450 FAD nmoles/mg protein RATS control phénobarbital pretreatment MICE C57BL/6J DBA/2J

RATIO P-450/FMN

0.71

0.075

0.15

2.1

0.08

0.13

26.3

0.51 0.53

0.021 0.013

0.094 0.090

24.3 40.8

9.5

Recently i n t e r e s t has developed i n the area of membrane s t r u c t u r e and the s p a t i a l r e l a t i o n s h i p of t h i s f l a v o p r o t e i n to cytochrome P-450 (49). A c o n s i d e r a t i o n of the stoichiometry (Table I ) of the f l a v o p r o t e i n to cytochrome P-450 r e v e a l s a s u r f e i t of hemeprotein molecules r e l a t i v e to i t s e l e c t r o n donating partner. The problem becomes even more complex when c o n s i d e r i n g the amphipathic p r o p e r t i e s of the f l a v o p r o t e i n and i t s p h y s i c a l l o c a t i o n on the surface of the membrane (50-54). The p o t e n t i a l r o l e of membrane f l u i d i t y f a c i l i t a t i n g the " p i n b a l l " l i k e m o t i l i t y of the f l a v o p r o t e i n as i t s e r v i c e s the dispersed pool of hemeprotein molecules i s a hypothesis (55, 56) which stands i n c o n t r a s t to the proposal (49,57-59) of c l u s t e r s or patches of multienzyme complexes based on hydrophobic i n t e r a c t i o n s i n the membrane. C l e a r l y , a more d e t a i l e d understanding o f membrane s t r u c t u r e i s a p r e r e q u i s i t e to the d e l i n e a t i o n of the process of r e d u c t i o n of the high s p i n form of o x i d i z e d cytochrome P-450 bound by substrate. R

Figure 9

Reaction with Carbon Monoxide. Reduced cytochrome P-450 r e a c t s r a p i d l y with carbon monoxide (Figure 9) to form a complex which serves as the hallmark f o r t h i s c l a s s of c e l l u l a r pigment (60-62). A c h a r a c t e r i s t i c absorbance band (Figure 10)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

160

XENOBIOTIC METABOLISM

at about 450 nm (hence the name P-450) i s a s s o c i a t e d w i t h t h i s complex. The chemical b a s i s f o r the unique absorbance band a t 450 nm f o r the carbon monoxide adduct of the reduced hemeprotein i s unknown although numerous s t u d i e s have been c a r r i e d out, i n p a r t i c u l a r using model compound complexes of heme, which suggest that a form of s u l f u r p a r t i c i p a t e s as one of the l i g a n d s of the heme of cytochrome P-450 (25-28) and that t h i s " s o f t l i g a n d " may i n p a r t be r e s p o n s i b l e f o r t h i s unusual property of t h i s c l a s s of hemeproteins. The bathochromic s h i f t i n absorbance of reduced cytochrome P-450, when i t r e a c t s w i t h carbon monoxide, stands i n sharp c o n t r a s t to other known hemeproteins where a hypsochromic s p e c t r a l s h i f t i s observed ( 6 ) . The a b i l i t y of reduced cytochrome P-450 to form complexes w i t h absorbance bands i n the v i c i n i t y of 450 nm i s not r e s t r i c t e d to i t s a c t i o n of reduced cytochrom t i v e , metyrapone, r e s u l t s i n the formation of an absorbance a t about 445 nm (63). Recently, i t was shown that a number of other organic compounds (such as s a f r o l e , the c l a s s of amphetamines, SKF-525A, e t c . ) r e a c t with reduced cytochrome P-450 to form d e r i v a t i v e s , c a l l e d product adducts or m e t a b o l i t e complexes, with absorbance bands i n the s p e c t r a l r e g i o n from 450 to 460 nm (64). The d e t a i l s of the chemistry of these types of r e a c t i o n s remains to be f u r t h e r s t u d i e d . S u f f i c e i t to say that a l l of these s t u d i e s s t r o n g l y suggest the existance of an environment i n the heme pocket of cytochrome P-450 which i s a t y p i c a l of other known hemeproteins (59).

448

0.2 —(

Figure 10. Optical absorbance properties of the carbon monoxide complex of reduced Cytochrome P-450. Cytochrome P-450 associated with liver microsomes prepared from animals pretreated with phénobarbital (PB) or 3-methylchohnthrene (3-MC) as well as Cytochrome P-450 isohted and purified from Pseudomonas putida (camphor) were reduced by sodium dithionite and examined by difference spectrophotometry. After gassing the contents of the sample cuvette with carbon monoxide, the various spectra were recorded. Differences in the location of the absorbance band maxima are indicated.

ο 0.0

-0.H

- 0 . 2 H 1

1

I

1

400

1

]—ι—ι—ι—r 4 50

Wavelength (nm)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5.

ESTABROOK ET A L .

Type I

161

Metabolism

Although the most commonly observed property f o r the reduced cytochrome P-450 complex w i t h carbon monoxide i s an absorbance band w i t h a maximum a t 450 nm, the exact locus of t h i s absorbance maximum can be s u b t l y s h i f t e d depending on the source of the hemeprotein. As shown i n F i g u r e 10, the absor­ bance band maximum i s l o c a t e d at about 446 nm f o r the cytochrome P-450 i s o l a t e d and p u r i f i e d from the camphor grown bacterium, Pseudomonas p u t i d a (43,65). The hemeprotein formed i n l i v e r microsomes as a r e s u l t of exposure of animals to p o l y c y c l i c hydrocarbons such as 3-methylcholanthrene has an absorbance band at 448 nm (66). These d i f f e r e n c e s undoubtedly r e s u l t from changes i n the environment of the heme f o r each of these hemep r o t e i n s as a consequence of s y n t h e s i s of p r o t e i n s with m o d i f i e d amino a c i d composition has been a p l e t h e r a of which f r e q u e n t l y confuse i n d i v i d u a l s not f a m i l i a r with the v a g a r i e s of a developing nomenclature which s t i l l seeks a common base.

1.0 4-

Λ

ι \

Science

0.54-

Figure 11. Photochemical action spec­ trum for the light reversibility of carbon monoxide inhibition of Cytochrome P450 catalyzed reactions.

1

400

Η

1

h

480 440 Wavelength (ητψ)

Rat liver microsomes were incubated with (Φ), codeine; (O), aminopyrine; or (Δ), acetanilide with various mixtures of carbon monoxide and oxygen and NADPH and irradiated with light of selected wavelengths (69j.

The property of reduced cytochrome P-450 r e a c t i n g w i t h carbon monoxide served as the primary c h a r a c t e r i s t i c f o r d e l i n e a t i n g the f u n c t i o n of t h i s hemeprotein i n the o x i d a t i v e metabolism of many d i f f e r e n t s u b s t r a t e s (68,69). L i k e other hemeproteins, the r e a c t i o n of carbon monoxide w i t h the reduced pigment i s an e q u i l i b r i u m r e a c t i o n which i s p h o t o s e n s i t i v e (70). The f i r s t d e f i n i t i o n of a b i o l o g i c a l f u n c t i o n f o r c y t o ­ chrome P-450 was achieved by a p p l y i n g the photochemical a c t i o n spectrum methodology of Warburg during a study of the 21 h y d r o x y l a t i o n of progesterone as c a t a l y z e d by the microsomal f r a c t i o n i s o l a t e d from the a d r e n a l cortex (68). Extension of these s t u d i e s to an examination of many mixed f u n c t i o n o x i d a t i o n r e a c t i o n s c a t a l y z e d by l i v e r microsomes (Figure 11)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

162

XENOBIOTIC M E T A B O L I S M

confirmed the general r o l e of t h i s hemeprotein i n the oxygen a c t i v a t i o n r e q u i r e d f o r the o x i d a t i v e conversion of a broad spectrum o f organic compounds. The f a c t that the carbon monoxide complex of reduced cytochrome P-450 has an absorbance band i n the Soret r e g i o n of the spectrum, which i s s i g n i f i c a n t l y d i s p l a c e d from the absorbance bands of comparable complexes of other reduced hemeproteins, serves as a u s e f u l property i n e v a l u a t i n g s p e c t r o p h o t o m e t r i c a l l y changes i n the c e l l u l a r content of t h i s pigment f o l l o w i n g exposure of animals to a v a r i e t y of chemicals. As shown i n F i g u r e 12, treatment of animals with a b a r b i t u r a t e , phénobarbital, r e s u l t s i n a marked i n c r e a s e i n the content of cytochrome P-450 i n the l i v e r of these animals. T h i s property of "enzyme i n d u c t i o n " can be a c c u r a t e l y and r a p i d l y monitored spectrophotometrically type presented i n F i g u r

HOURS Figure 12. Induction of liver microsomal Cytochrome P-450 following treatment of rats with phénobarbital. Male rats (150-200 gm) were injected intraperitoneally daily with 80 mg of phénobarbital /kg body weight. Animals were sacrificed at the times indicated and the content of Cytochrome P-450 in (O O), isolated liver microsomes as well as ( Ο — Ο ), liver homogenates (IS).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5.

ESTABROOK ET A L .

Type

I

Metabolism

163

cytochrome P-450 i n an organ such as the l i v e r . Subsequent to treatment of the animals f o r f i v e days w i t h phénobarbital, approximately 15 percent of the p r o t e i n of the endoplasmic r e t i c u l u m of l i v e r i s composed of t h i s one c l a s s of p r o t e i n (71). Likewise, as much as 5 percent of the p r o t e i n of the l i v e r can be cytochrome P-450. T r u l y , cytochrome P-450 i s not a minor c o n s t i t u e n t i n t h i s and other organs - i t i s the dominant species of hemeprotein which i s present a t a c o n c e n t r a t i o n e q u i v a l e n t to that of myoglobin i n some muscle t i s s u e .

R

+2

Fe -R

Figure 13

Reaction w i t h Oxygen. In the presence of molecular oxygen reduced cytochrome P-450 r e a c t s r a p i d l y to form an intermediate (Figure 13) termed oxycytochrome P-450. The e x i s t e n c e of an oxygenated form of reduced cytochrome P-450 was p r e d i c t e d from the e a r l y experiments designed to determine those f a c t o r s which i n f l u e n c e the extent of carbon monoxide i n h i b i t i o n of cytochrome P-450 c a t a l y z e d r e a c t i o n s (68). These s t u d i e s had c l e a r l y shown that carbon monoxide was an i n h i b i t o r which was competit i v e w i t h oxygen, i . e . , the magnitude of i n h i b i t i o n observed i s d i c t a t e d by the r a t i o of carbon monoxide to oxygen r a t h e r than simply the c o n c e n t r a t i o n of carbon monoxide i n the r e a c t i o n system. Studies by Ishimura £t a l . (72,73) as w e l l as Gunsalus et a l . (74,75), using the s o l u b l e and p u r i f i e d cytochrome P-450 i s o l a t e d from Pseudomonas p u t i d a , c l e a r l y demonstrated the presence of a reasonably s t a b l e d e r i v a t i v e of the reduced hemeprotein when exposed to oxygen. T h i s complex was i d e n t i f i a b l e s p e c t r o p h o t o m e t r i c a l l y , as shown i n F i g u r e 14, and

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

164

^

1 400

ι

1

1

500

1 600

1

Γ 700

Wavelength (nm) Figure 14. Absorption spectra of oxidized, reduced, and Oxycytochrome P-450. Samples of purified Cytochrome P-450 isolated from Pseudomonas putida were examined spectrophotometrically in the presence of the substrate camphor (73).

WAVELENGTH(nm)

Figure 15. Repetitive scan difference spectral measurements of liver microsomal Cytochrome P-450 during the NADPH supported steady state metabolism of hexobarbital. Liver microsomes from phénobarbital treated male rats were diluted to 1 mg of protein/mL in a buffer mixture containing 50mM TRIS chloride (pH 7.5), 150mM KCl, 10 mM MgCk, and 2mM hexobarbital. NADPH (0.5mM final concentration) was added to initiate the reaction and the difference spectra recorded every 30 sec. The absorbance band at about 440 nm is attributed to Oxycytochrome P-450.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5.

ESTABROOK

χ. F e i

°2

Type

ET AL.

X-Fe

+2

l

-

°i

+3

I

165

Metabolism

• X· Fe

+3

+ 01 r SOD

H 0 2

2

Academic Press Figure 16.

Possible valence states of Oxycytochrome P-450 and its dissociation to form superoxide

was recognized to resemble the oxygenated form o f other hemep r o t e i n s (76-79). The a b i l i t y to demonstrate the presence o f oxycytochrome P-450 during the f u n c t i o n i n g of the i n t e g r a t e d and membrane bound e l e c t r o n transport system of microsomes (the C l a s s Β type of cytochrome P-450) i s more complex and subject to many v a r i a b l e s . R e p e t i t i v e scan spectrophotometric measure­ ments during the NADPH supported aerobic steady s t a t e o x i d a t i o n of a v a r i e t y o f substrates by l i v e r microsomes r e v e a l s (Figure 15) the presence o f an absorbance band with a r a t h e r broad maximum a t about 440 nm i n the d i f f e r e n c e spectrum (80,81). This absorbance band i s s i m i l a r to the s p e c t r a l intermediate seen with the p u r i f i e d cytochrome P-450 from Pseudomonas putida. The magnitude o f t h i s absorbance band of oxycytochrome P-450, when studied using l i v e r microsomes, i s markedly i n ­ fluenced by the type o f substrate studied, the a s s o c i a t e d a c t i v i t y of NADPH-cytochrome P-450 reductase, i . e . , the balance of a d d i t i o n of the f i r s t e l e c t r o n versus the second e l e c t r o n (see below) f o r the f u n c t i o n of cytochrome P-450, and the presence of excess NADPH and oxygen. As shown i n Figure 15, the concentration of oxycytochrome P-450 p r o g r e s s i v e l y de­ creases as the oxygen concentration i n the r e a c t i o n medium decreases. Oxycytochrome P-450 can be considered to be an e q u i l i b r i u m between two ternary complexes. In one case (Figure 16), the f e r r o u s hemeprotein can be envisioned as complexed with a molecule o f superoxide. The l a t t e r can be v i s u a l i z e d as de­ caying g i v i n g r i s e to the high s p i n f e r r i c hemeprotein sub­ s t r a t e complex and the superoxide anion (or i t s protonated form, the perhydroxyl r a d i c a l ) . The presence of a d v e n t i t i o u s superoxide dismutase a s s o c i a t e d with the microsomal f r a c t i o n

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would then c a t a l y z e the formation of hydrogen peroxide. It i s now w e l l e s t a b l i s h e d that hydrogen peroxide i s formed during NADPH o x i d a t i o n by l i v e r microsomes (82,83). Although the source of t h i s hydrogen peroxide remains s p e c u l a t i v e , the decomposition of oxycytochrome P-450 seems a very l i k e l y possi b i l i t y and recent s t u d i e s (83-85) have supported the hypothesis that superoxide i s generated during the f u n c t i o n of cytochrome P-450. Thus, one can consider an "oxidase type" c y c l e f o r cytochrome P-450 (Figure 17) whereby e l e c t r o n s o r i g i n a t i n g from NADPH are d i v e r t e d to the r e d u c t i o n of oxygen f o r the formation of hydrogen peroxide r a t h e r than f o r the mixed f u n c t i o n o x i d a t i v e metabolism of x e n o b i o t i c s . In v i t r o s t u d i e s using i s o l a t e d l i v e r microsomes have revealed the a b i l i t y of many compounds to s t i m u l a t e the r a t e of formation of hydrogen peroxide formation concomitan tochrome P-450 (86,87) genated hydrocarbons (88,89) i t i s apparent that a s i g n i f i c a n t d i v e r s i o n of e l e c t r o n s occurs r e s u l t i n g i n an uncoupling of R

Figure 17.

Dissociation of superoxide from Oxycytochrome F-450 for the formation of hydrogen peroxide

oxygenation r e a c t i o n s . T h i s may be of p a r t i c u l a r i n t e r e s t to those concerned w i t h p e s t i c i d e s because of the frequent use of compounds, such as Lindane. The Second E l e c t r o n . Oxycytochrome P-450, to which a molecule of s u b s t r a t e i s bound, undergoes f u r t h e r r e d u c t i o n to an intermediate termed peroxycytochrome P-450 (Figure 18). L i k e oxycytochrome P-450, the proposed intermediate peroxycytochrome P-450 can be w r i t t e n i n a v a r i e t y of equivalent e l e c t r o n valence forms. The i n a b i l i t y to i s o l a t e and chara c t e r i z e t h i s proposed intermediate has impeded f u r t h e r understanding of i t s chemistry and p h y s i c a l p r o p e r t i e s

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5.

ESTABROOK ET A L .

Type

I

Metabolism

167

R

Fe +3

X

e

Fe -R +2

0

2

Τ e

Figure 18

although i t s e x i s t e n c e as a t r a n s i e n t intermediate i n the f u n c t i o n o f cytochrome P-450 has been g e n e r a l l y assumed. The s t o i c h i o m e t r y of mixed f u n c t i o n o x i d a t i o n r e a c t i o n s r e q u i r e s the p a r t i c i p a t i o n of two e l e c t r o n s ; the r e d u c t i o n of the f e r r i c cytochrome P-450 complex has been e s t a b l i s h e d (47,48) to be a one e l e c t r o n t r a n s f e r r e a c t i o n . Studies with the p u r i f i e d b a c t e r i a l cytochrome P-450 have shown (7) the transformation of oxycytochrome P-450 by the a d d i t i o n of the reduced i r o n s u l f u r p r o t e i n , p u t i d a r e d o x i n . F u r t h e r , the a b i l i t y to observe s p e c t r o p h o t o m e t r i c a l l y intermediates formed during the r e a c t i o n of f e r r i c cytochrome P-450 with organic hydroperoxides, such as cumene hydroperoxide, which d i f f e r from oxycytochrome P-450 s u b s t a n t i a t e s the e x i s t e n c e of a d d i t i o n a l oxygen c o n t a i n i n g complexes of cytochrome P-450 (90). P r o t o n a t i o n of peroxycytochrome P-450 and the d i r e c t r e l e a s e of hydrogen peroxide concomitant with the formation of the f e r r i c cytochrome P-450 complex with s u b s t r a t e (Figure 19) i s an a l t e r n a t i v e means of e x p l a i n i n g the formation of hydrogen peroxide during NADPH o x i d a t i o n by l i v e r microsomes. Consid­ e r a b l e work has centered on d i s t i n g u i s h i n g the two a l t e r n a t i v e s f o r the formation of hydrogen peroxide, i . e . , the decomposition of oxycytochrome P-450 with the r e l e a s e of superoxide which then undergoes dismutation or the p r o t o n a t i o n of peroxycytochrome P-450 and the d i r e c t formation of hydrogen peroxide. An examination of the r e a c t i o n scheme shown i n F i g u r e 19 r e v e a l s a major d i f f e r e n c e between these two options which should d e l i n ­ eate the dominant pathway f o r hydrogen peroxide formation. As discussed above, two e l e c t r o n t r a n s f e r steps are r e q u i r e d to form peroxycytochrome P-450 while only one e l e c t r o n t r a n s f e r

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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step i s needed to generate oxycytochrome P-450. Previous s t u d i e s of drug metabolism by the cytochrome P-450 c o n t a i n i n g l i v e r microsomes revealed a " s y n e r g i s t i c e f f e c t " of NADH

R

e Figure 19.

Proposed formation of hydrogen peroxide from Peroxycytochrome P-450

X I NADH

Figure 20. Scheme showing the proposed role of reduced Cytochrome h as the donor of the electron required for the reduction of Oxycytochrome P-450 and the formation of Per oxycytochrome P-450 5

when supplementing the NADPH dependent mixed f u n c t i o n o x i d a t i o n of v a r i o u s s u b s t r a t e s . This " s y n e r g i s t i c e f f e c t " e l i c i t e d by NADH (91-94) was a t t r i b u t e d to a r o l e f o r reduced cytochrome b^ as the donor of the second e l e c t r o n r e q u i r e d f o r the c y c l i c f u n c t i o n of cytochrome P-450. This i s i l l u s t r a t e d i n F i g u r e 20. The presence of NADH would spare reducing e q u i v a l e n t s o r i g i n a t i n g from NADPH needed f o r the r e d u c t i o n of

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5. ESTABROOK E T A L .

Type

I

Metabolism

169

cytochrome b^ and i t would a l s o i n c r e a s e the extent of steady s t a t e r e d u c t i o n of t h i s hemeprotein. Comparable s t u d i e s were c a r r i e d out to determine whether a " s y n e r g i s t i c e f f e c t " by NADH on the NADPH dependent formation of hydrogen peroxide a l s o occurred. These r e s u l t s were negative (83,85). This suggests that the p r i n c i p a l pathway f o r the formation of R

ι I e

Figure 21. Proposed formation of an oxene-type intermediate during the cyclic function of Cytochrome P-450.

hydrogen peroxide was the decay of oxycytochrome P-450 r a t h e r than the p r o t o n a t i o n of peroxycytochrome P-450. Obviously, more d e f i n i t i v e experiments w i l l have to be c a r r i e d out but the present l i m i t a t i o n i n methodologies precludes the design of such unequivacol s t u d i e s . Other P o s s i b l e Intermediates. The sequence of r e a c t i o n s i n v o l v e d i n the " a c t i v a t i o n of oxygen" from peroxycytochrome P-450 a r e unknown. Much has been w r i t t e n and many s p e c u l a t i v e hypotheses have been proposed but no experimental v e r i f i c a t i o n e x i s t s to b e t t e r d e f i n e p o s s i b l e intermediates. One such hypothesis, which i s very a t t r a c t i v e , proposes the formation of an oxene complex of cytochrome P-450 r e s u l t i n g from the p r o t o n a t i o n of peroxycytochrome P-450 and the r e l e a s e of a molecule of water (95,96). This i s i l l u s t r a t e d i n F i g u r e 21. The e x i s t e n c e of an oxenoid species of oxygen would f u l f i l l the requirement of a h i g h l y e l e c t r o p h i l i c atom which could p a r t i c i p a t e i n h y d r o x y l a t i o n r e a c t i o n s . Recently, the p r e s ­ ence of f r e e - r a d i c a l species has a l s o been deduced from deuterium i s o t o p e experiments (97). However, the f a i l u r e to detect any measureable c o n c e n t r a t i o n of f r e e r a d i c a l s during the microsomal c a t a l y z e d o x i d a t i v e metabolism of any s u b s t r a t e yet examined i n d i c a t e s the absence of a steady s t a t e concen­ t r a t i o n of such proposed f r e e r a d i c a l s . Additional inter­ mediates such as the carbanion form of some s u b s t r a t e s , the

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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e x i s t e n c e of a carbene form of the s u b s t r a t e , and the generat i o n of epoxides should a l l be considered as p o s s i b i l i t i e s u n t i l our l e v e l of knowledge i s s u f f i c i e n t l y expanded to permit choices based on experimental r a t h e r than t h e o r e t i c a l considerations. The

Role of the Membrane

Cytochrome P-450 present i n mammalian t i s s u e s i s recogn i z e d to be i n t i m a t e l y a s s o c i a t e d with membranes. Studies of membrane bound enzymes have the disadvantage that s p e c i a l methodologies, such as the use of d i f f e r e n c e spectrophotometry as described e a r l i e r , as w e l l as the presence of high concent r a t i o n s of l i p i d can introduc v a r i a b l e that difficult to f u l l y evaluate. Th can perturb the p a t t e r sequestering of the h i g h l y l i p o p h i l i c substrates that are o x i d a t i v e l y transformed during the f u n c t i o n of cytochrome P450 (31,98). Recently considerable i n t e r e s t has centered on the s p a t i a l and s t r u c t u r a l o r g a n i z a t i o n of cytochrome P-450 and i t s a s s o c i a t e d e l e c t r o n t r a n s f e r p r o t e i n s w i t h i n the mileau of the microsomal membrane. The unique stoichiometry of f l a v o p r o t e i n s and cytochromes ( c f . Table I) have provided the opportunity to evaluate the r o l e of the membrane i n regul a t i n g and modifying the types of r e a c t i o n s c a t a l y z e d and the i n f l u e n c e of r e s t r i c t e d m o b i l i t y of p r o t e i n w i t h i n the l i p i d mosaic of the membrane. I t has been proposed (49,58,59) that cytochrome P-450 e x i s t s as c l u s t e r s (Figure 22) which

K" 200A—H Figure 22. Schematic of the microsomal membrane showing clusters of molecules of Cytochrome P-450 surrounding the flavoprotein reductase

can form patches of e l e c t r o n t r a n s p o r t complexes w i t h i n or withon the membrane. Much controversy surrounds t h i s i n t e r -

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5.

ESTABROOK ET A L .

Type I

Metabolism

p r e t a t i o n (55,56) and the extent of membrane f l u i d i t y , as i t i n f l u e n c e s the cytochrome P-450 dependent metabolism of subs t r a t e s , remains as an area r e q u i r i n g f u r t h e r study. The s o l u t i o n of t h i s i n t r i g u i n g problem w i l l undoubtedly i n f l u e n c e our f u r t h e r understanding of the r e a c t i o n s i n v o l v e d and the r e l a t i o n s h i p of s t u d i e s c a r r i e d out i n v i v o to those that have been experimentally a s c e r t a i n e d by i n v i t r o s t u d i e s . The importance of the membrane to s t u d i e s of the metabolism of x e n o b i o t i c s cannot be underestimated. Most of the compounds which are o x i d a t i v e l y transformed are h i g h l y l i p o p h i l i c . Further, the l i p i d composition of the microsomal membrane can be r e a d i l y modified by the type of d i e t employed as w e l l as the age and sex of the experimental animal system under study. M u l t i p l e Types of Cytochrom Any c o n s i d e r a t i o n of the f a c t o r s which i n f l u e n c e the r o l e of cytochrome P-450 i n the metabolism of x e n o b i o t i c s must account f o r the ever i n c r e a s i n g body of evidence which shows the m u l t i p l e types of cytochrome P-450 that e x i s t i n a s i n g l e c e l l type, such as the hepatocyte. I t has been known f o r many years that v a r i o u s inducing agents can cause a p r e f e r e n t i a l s t i m u l a t i o n i n the metabolism of drug s u b s t r a t e s . The b a s i s f o r t h i s d i f f e r e n c e was c l e a r l y d e l i n e a t e d by the observation (66) that treatment of animals with the inducing agent, 3methylcholanthrene, r e s u l t e d i n the s y n t h e s i s of a hemeprotein a s s o c i a t e d with l i v e r microsomes which had o p t i c a l absorbance p r o p e r t i e s d i s t i n c t from a s i m i l a r pigment induced upon t r e a t ment of animals with phénobarbital ( c f . Figure 10). The l a s t decade has seen major advances i n the i s o l a t i o n and p u r i f i c a t i o n of d i f f e r e n t types of cytochromes P-450. D i f ferences i n response to s p e c i f i c a n t i b o d i e s , p r o t e i n s t r u c t u r e , and molecular weights are a l l subjects of current research (99-104). A d i r e c t demonstration of the change i n the p a t t e r n of types of cytochrome P-450 a s s o c i a t e d with l i v e r microsomes i s o l a t e d from animals subjected to d i f f e r e n t inducing agents i s shown by the polyacrylamide g e l e l e c t r o p h o r e s i s r e s u l t s presented i n F i g u r e 23. Dramatic a l t e r a t i o n s i n the magnitude as w e l l as m o b i l i t y of p r o t e i n s i n the molecular weight range between 45,000 and 50,000 can be seen a f t e r exposure of animals to the inducing agents, phénobarbital or pregnenolong-16alpha-carbonitrile. Thus one must consider, when d i s c u s s i n g the i n v i v o enzymatic a c t i v i t y of cytochrome P-450, not only the i n f l u e n c e of the membrane and the a v a i l a b i l i t y of cof a c t o r s such as reduced p y r i d i n e n u c l e o t i d e s and oxygen but a l s o the presence of unique types of cytochrome P-450. A l o g i c a l extension of these s t u d i e s i s to p o s t u l a t e the presence of unique types of cytochrome P-450 which possess a s p e c i f i c i t y f o r v a r i o u s c l a s s e s of s u b s t r a t e s . As of t h i s w r i t i n g , the demonstration of s u b s t r a t e s p e c i f i c i t y has not

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171

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XENOBIOTIC METABOLISM

Figure 23. PAGE patterns of proteins present in liver microsomes from untreated animals (control), pregnenolone-16 a-carbonitrile (PCN)-, or phénobarbital (PB)treated male rats. Microsomes were isolated and treated with SDS prior to electrophoresis and staining with Coomassie blue.

p r e t r e a t e d with a p o l y c y c l i c hydrocarbon has a high a c t i v i t y i n the metabolism of benzo(a)pyrene but a l s o c a t a l y z e s the o x i d a t i v e metabolism of drugs such as benzphetamine. This puzzle becomes even more i n t r i g u i n g when e v a l u a t i n g the recognized competitive e f f e c t s of v a r i o u s types of substrates when r a t e s of metabolism a r e measured using the membrane bound form of the enzyme. C l e a r l y , the i n t e g r a t i o n of knowledge gained from s t u d i e s with v a r i o u s types of p u r i f i e d cytochrome P-450 as w e l l as the e l e c t r o n transport complexes e x i s t e n t i n the membrane w i l l be r e q u i r e d before a meaningful assessment of the i n v i v o transformation of x e n o b i o t i c s can be accomplished. Concluding Remarks The d e l i n e a t i o n of the sequence o f events which occurs as the hemeprotein, cytochrome P-450, r e a c t s s e q u e n t i a l l y with a molecule of s u b s t r a t e to be metabolized, an e l e c t r o n d e r i v e d from NADPH and t r a n s f e r r e d v i a the f l a v o p r o t e i n , NADPH-cytochrome P-450 reductase, a molecule of oxygen, and then a second e l e c t r o n has been b r i e f l y discussed i n the foregoing s e c t i o n s .

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

5.

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Type

I

Metabolism

173

The r e s u l t i n g o v e r a l l scheme shown i n F i g u r e 24 attempts to s y n t h e s i z e our present understanding of t h i s c y c l i c process. One must r e c o g n i z e , however, that much remains to be r e v e a l e d i f we a r e to g a i n s u f f i c i e n t knowledge which may permit the development of agents that can p r e d i c t a b l y perturb t h i s

Figure 24. Scheme of intermediates formed during the cyclic reaction of Cytochrome P-450 associated with the metabolism of many xenobiotics. The possible sites of formation of hydrogen peroxide as well as the pathway of metabolism initiated by organic hydroperoxides are illustrated.

r e a c t i o n system. Indeed, achievement of t h i s knowledge i s a necessary p r e r e q u i s i t e to attainment of the goal of p r e d i c t i n g the o x i d a t i v e transformation of x e n o b i o t i c s to h i g h l y t o x i c agents (many w i t h the known p o t e n t i a l of s e r v i n g as carcinogens) or to products that a r e not d e t r i m e n t a l to c e l l u l a r f u n c t i o n . The i n v e n t o r y of questions which remain unanswered i s long and i n c l u d e s such items as f o l l o w s : a) What i s " a c t i v e oxygen" and how does i t perform the necessary chemistry to i n t e r a c t with such a broad spectrum of chemicals as those known to be metabolized by c y t o chrome P-450? b) What i s the unique chemistry of cytochrome P-450 that permits i t to r e a c t with oxygen and the s u b s t r a t e molec u l e s i n a manner that permits the c a t a l y s i s of x e n o b i o t i c transformation?

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c) Do the m u l t i p l e forms of cytochrome P-450, which are s e l e c t i v e l y induced by v a r i o u s chemicals, r e f l e c t more than a s i n g l e mechanism f o r the a c t i v a t i o n of oxygen? d) What c o n s t r a i n t s are imposed by the hydrophobic environs of the membrane and a s s o c i a t e d problems of p r o t e i n p o l a r i t y , f l u i d i t y , or existence as c l u s t e r s ? e) What f a c t o r s d i c t a t e the p a t t e r n of e l e c t r o n t r a n s p o r t from reduced p y r i d i n e n u c l e o t i d e s and how are these r e f l e c t e d i n the case of s u b s t r a t e metabolism? f ) Is the concomitant formation of hydrogen peroxide, known to occur during the cytochrome P-450 metabolism of s u b s t r a t e s , of an These and many more questions are under i n t e n s i v e i n v e s t i g a t i o n i n many l a b o r a t o r i e s . Undoubtedly, new r e s u l t s w i l l r e v e a l the f r a g i l e foundation on which our current hypotheses are developed. The importance of knowning how t h i s enzyme system f u n c t i o n s i s at the f o r e f r o n t of s c i e n t i f i c concern during the present time when a great d e a l of a t t e n t i o n has been focused on the wide spread d i s s e m i n a t i o n i n our environment of xenobiotics. The f u t u r e holds great promise to b r i n g f o r t h new ideas and new concepts. I t w i l l be i n t e r e s t i n g to r e f l e c t back on the meager beginning where we stand now.

Abstract The oxidative conversion of many lipophilic chemicals which are xenobiotics occurs by means of an enzyme system where the hemeprotein, cytochrome P-450, functions as an oxygenase. The importance of these types of reactions is now recognized as the primary step in the metabolic formation of many toxic agents including carcinogens. The details of our current understanding of cytochrome P-450 interaction with the organic substrate molecule, oxygen, and electrons derived from reduced pyridine nucleotides is described in the present paper. Particular attention is directed toward the sequence of reactions occurring and the attendent problems of defining the parameters which regulate the functionality of the membrane bound enzyme complex. The methodologies employed for studies to define the role of cytochrome P-450 in the metabolism of drugs, steroids, and chemicals which can be converted to carcinogens, have been described and typical results illustrated. The extension of these studies to better define the specificity of the reaction system as well as the properties of reactive intermediates poses the direction for future experimentation.

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RECEIVED December 20, 1978.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

6 The Use of Animal Subcellular Fractions to Study Type II Metabolism of Xenobiotics MAUREEN

J. C R A W F O R D and D A V I D H . H U T S O N

Shell Research Ltd., Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent, M E 9 8AG, U . K .

Type II biotransformations or conjugation reactions are enzyme­ -catalysed energy-requiring biosyntheses. The ultimate reaction requires a high-energy dono and an appropriate transferase contain the endogenous conjugating agent (conjugand) (e.g. glucuronic acid, as in UDPGA) or i t may contain the xenobiotic (e.g. 4-chlorobenzoic acid, as in 4-chlorobenzoyl-CoA). If the xenobiotic is to be activated to become a donor substrate, other enzymes and high energy substrates (e.g. ATP) are called into action. If we assume for the moment that the low molecular weight substrates for these reactions have equal access to every component in the cell, then the subcellular location of the transferring enzyme will be the controlling factor in the subcellular distribution of the conjugation reaction. History of the Technique The metabolism of foreign compounds has been studied in various subcellular fractions from about 1950. Brodie and coworkers (1) presented the first overview of the enzyme­ -catalysed metabolism of these compounds in 1958. In the intervening years, the properties of microsomes and other subcellular fractions in relation to the metabolism and toxicity of drugs, pesticides and, more recently, 'environmental' chemicals have received an enormous amount of study. However, i t is interesting to note that the best of the earlier reviews of the enzymology of foreign compound metabolism by J. R. Gillette in 1963 (2) contains the essential details of each of the conjugation reactions referred to below. Progress has not been even: i t has proved difficult to keep our treatment of glucuronyltransferase within reasonable limits, yet amino acid conjugation, despite some very interesting species differences, has received very little attention at the enzyme level. For our practical purposes, the animal cell can be regarded as composed of nucleus, endoplasmic reticulum, mitochondria 0-8412-0486-l/79/47-097-181$12.25/0 © 1979 A m e r i c a n C h e m i c a l Society

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and lysosomes suspended together i n water c o n t a i n i n g d i s s o l v e d p r o t e i n s and small molecules. One o f the values o f studying type I I r e a c t i o n s at the s u b c e l l u l a r l e v e l l i e s i n the informa t i o n gained about the r o l e of these components and the mechanism of the r e a c t i o n s . When the c e l l i s fragmented and the v a r i o u s o r g a n e l l e s are separated, the conjugation r e a c t i o n under i n v e s t i g a t i o n may not be observed i n any o f the f r a c t i o n s because enzymes have been separated from mandatory c o f a c t o r s . For example, g l u c u r o n i d a t i o n r e a c t i o n s are not observed i n the i s o l a t e d endoplasmic r e t i c u l u m (which contains the t r a n s f e r a s e ) because the high-energy donor s u b s t r a t e , u r i d i n e diphosphog l u c u r o n i c a c i d (UDPGA), i s l o c a t e d i n the s o l u b l e f r a c t i o n . Thus the e f f o r t r e q u i r e d to r e c o n s t i t u t e the conjugation r e a c t i o n s at the s u b c e l l u l a r l e v e l has been v i t a l to the f u l l understanding of t h e i r mechanisms None of t h i s coul biochemists who have developed an understanding of intermediary metabolism and s u b c e l l u l a r f r a c t i o n a t i o n procedures. D i f f e r e n t i a l c e n t r i f u g a t i o n i s s t i l l the main method used and t h e r e f o r e the steady development of p r e p a r a t i v e - s c a l e c e n t r i f u g e s has a l s o been very important. The

Preparation

of S u b c e l l u l a r F r a c t i o n s

S u b c e l l u l a r f r a c t i o n a t i o n i s u s u a l l y c a r r i e d out by d i f f e r e n t i a l c e n t r i f u g a t i o n . However, other methods such as p r e c i p i t a t i o n and chromatography have been i n v e s t i g a t e d f o r the i s o l a t i o n of s p e c i f i c f r a c t i o n s . 2.1 D i f f e r e n t i a l C e n t r i f u g a t i o n D i f f e r e n t i a l c e n t r i f u g a t i o n i s by f a r the most widely used technique. I t i s e f f e c t i v e , clean and gentle and although i t could have been d i s p l a c e d at one time by a f a s t e r technique, the a v a i l a b i l i t y of p r e p a r a t i v e u l t r a - c e n t r i f u g e s capable of up to 500,000 g has cut down p r e p a r a t i o n times to a very competitive l e v e l . The machines now a l s o operate at acceptable noise l e v e l s . Fresh t i s s u e i s homogenised i n b u f f e r e d s a l t s o l u t i o n and the r e s u l t a n t homogenate i s c e n t r i f u g e d at about 600 g to remove unbroken c e l l s and c e l l d e b r i s . The supernatant i s then c e n t r i fuged at 8000-10,000 g f o r about 20 min to sediment the mitochondria. Some lysosomes are sedimented at t h i s stage. The supernatant i s then c e n t r i f u g e d at about 200,000 g f o r 20 min to sediment the fragmented endoplasmic r e t i c u l u m (microsomal f r a c t i o n ) together with some lysosomes. The r e s u l t i n g supernatant i s the s o l u b l e f r a c t i o n ( c y t o s o l ) . Each of the p a r t i c u l a t e f r a c t i o n s may be f u r t h e r p u r i f i e d by washing and r e c e n t r i f u g a t i o n . They may be i n d i v i d u a l l y f u r t h e r p u r i f i e d by d e n s i t y gradient c e n t r i f u g a t i o n . The i s o l a t i o n o f microsomes has r e c e i v e d a l o t of a t t e n t i o n from drug metabolism researchers because of the importance of the microsomal mono-oxygenase system i n x e n o b i o t i c metabolism (see preceding Chapter). When

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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the r o u t i n e procedure o u t l i n e d above i s c a r r i e d out there i s i n e v i t a b l y some contamination of one f r a c t i o n by another. This i s best seen by c o n s i d e r i n g a f r a c t i o n a t i o n of r a t l i v e r c a r r i e d out i n our l a b o r a t o r y by Wright and coworkers (3) during s t u d i e s on the e f f e c t of the i n g e s t i o n of d i e l d r i n on hepatocytes. The f r a c t i o n a t i o n procedure was monitored by measuring the a c t i v i t i e s of s e v e r a l enzymes i n each f r a c t i o n , i n c l u d i n g s u c c i n i c dehydrogenase ( m i t o c h o n d r i a l ) , and glucose-6-phosphatase and c h l o r f e n vinphos d e a l k y l a s e (microsomal). Some of these r e s u l t s are i l l u s t r a t e d i n Figure 1. Often we r e q u i r e samples of only the microsomal and c y t o s o l i c f r a c t i o n s , i n which case we r o u t i n e l y prepare 20% homogenates i n 0.1 M potassium phosphate b u f f e r pH 7.4, c e n t r i fuge at 10,000 g f o r 20 min and use t h i s supernatant to prepare microsomes (190,000 g/3 (4). P r o t e i n concentration s p e c i f i c a c t i v i t i e s can be c a l c u l a t e d . These are measured u s i n g e i t h e r the s e n s i t i v e procedure d e s c r i b e s by Lowry et a l (5) or the simple m o d i f i c a t i o n of the b i u r e t r e a c t i o n d e s c r i b e d by Robinson and Hodgen (6). Precipitation Rapid methods f o r the p r e p a r a t i o n of microsomal f r a c t i o n s have been looked f o r i n recent y e a r s . I s o e l e c t r i c p r e c i p i t a t i o n of l i v e r microsomes from p o s t - m i t o c h o n d r i a l supernatant at pH 5.4 i s a u s e f u l such method (_7) . Mono-oxygenase c h a r a c t e r i s t i c s i n the product compare very w e l l with those of conventional microsomes but the status of the g l u c u r o n y l t r a n s f e r a s e was not reported. These preparations contain about 45% more p r o t e i n than do conventional preparations and t h e i r p o s s i b l e contamina t i o n by c y t o s o l enzymes, such as the g l u t a t h i o n e t r a n s f e r a s e s , r e q u i r e s f u r t h e r i n v e s t i g a t i o n . A calcium i o n sedimentation method was a c c i d e n t l y discovered during a p r e p a r a t i o n of plasma membranes which became contaminated with aggregated microsomes. The method was then developed s p e c i f i c a l l y f o r microsomes (8). Mono-oxygenase c h a r a c t e r i s t i c s were s i m i l a r to those i n conventionally-prepared microsomes but again g l u c u r o n y l t r a n s f e r a s e was not t e s t e d (9)(10). The method has been a p p l i e d to r a t and r a b b i t kidney and lung t i s s u e . Microsomal y i e l d s from the l a t t e r d i f f e r from those obtained by c e n t r i f u g a t i o n i n that there i s a higher y i e l d of p r o t e i n with lower s p e c i f i c a c t i v i t y ^

}

'

Gel F i l t r a t i o n Chromatography Microsomes and c y t o s o l of r a t l i v e r 13,000 g supernatant have been separated by g e l f i l t r a t i o n through Sepharose 2B; the microsomes were c o l l e c t e d i n the e x c l u s i o n volume (Vo) and the c y t o s o l , between Vo and Vt (12). The method has been a p p l i e d to r a t lung with remarkable success g i v i n g microsomes with very high s p e c i f i c a c t i v i t i e s ( f o r o x i d a t i v e r e a c t i o n s ) (13). An example of one of our own separations using t h i s technique i s shown i n Figure 2 (10 ml of 10,000 g supernatant from 40% r a t

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

184

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glucose—6— phosphatase

succinic dehydrogenase

I I

5 0

METABOLISM

chlorfenvinphos dealkylase

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- ϋ Distribution of marker enzymes between subcelluhr fractions

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CRAWFORD AND HUTSON

Type

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Protein mg.ml

-1

10

15 Fraction number

20

25

LL

cytochrome P450

Haemoglobin

2 Figure 2.

Gel filtration of rat liver 10,000 g supernatant on Sepharose 2B

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

30'

186

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l i v e r homogenate a p p l i e d to a 2.5 cm χ 25 cm column run at upward flow, 1 ml/min; 5 ml f r a c t i o n s c o l l e c t e d ) .

4-5°,

Advantages of the S u b c e l l u l a r F r a c t i o n s I n d i v i d u a l s u b c e l l u l a r f r a c t i o n s , supplemented with the appropriate c o f a c t o r s , u s u a l l y a f f o r d information on d i s c r e t e steps i n metabolic pathways i n a way that i s not p o s s i b l e u s i n g animals, organs or c e l l s . The study of c o f a c t o r requirements provides information on the mechanism of each r e a c t i o n , as do studies w i t h i s o l a t e d p u r i f i e d enzymes. The conditions f o r the r e a c t i o n to be studied can be optimised and f u r t h e r metabolism can, i f necessary, be blocked so that an intermediate of i n t e r e s t can be i s o l a t e d . In a converse sense mechanisms of the more conjugatio , y of the s u b c e l l u l a r l o c a t i o n and c o f a c t o r requirements of the b i o t r a n s f o r m a t i o n of a x e n o b i o t i c o f f e r s u s e f u l information on the metabolism of that compound. I t i s important to know each d i s c r e t e step i n the b i o t r a n s f o r m a t i o n of a x e n o b i o t i c because we then have a chance of i d e n t i f y i n g intermediates that may prove hazardous under c e r t a i n circumstances. I t i s important a l s o to know the e f f e c t of t o x i c a n t s on the f u n c t i o n of a p a r t i c u l a r c e l l o r g a n e l l e . This requires the study of s u b c e l l u l a r f r a c t i o n s . Another important advantage of s u b c e l l u l a r techniques i s that the f r a c t i o n s are r e l a t i v e l y easy to prepare and they are reasonably robust provided that c o r r e c t conditions are used. Conditions are described i n some d e t a i l i n the i n d i v i d u a l s e c t i o n s below. Disadvantages of the Technique The d i f f i c u l t i e s that may be experienced i n working with s u b c e l l u l a r f r a c t i o n s are d e t a i l e d below i n the sections on the i n d i v i d u a l conjugation r e a c t i o n s . However, some u s e f u l g e n e r a l ­ i s a t i o n s may be made. The e x t r a p o l a t i o n back to the s i t u a t i o n i n v i v o from s u b c e l l u l a r systems i s a long one. We tend to base our comparisons on enzyme a c t i v i t y measured under conditions favouring good k i n e t i c s (e.g. zero order f o r c o f a c t o r s , r a t e l i n e a r with time and p r o t e i n c o n c e n t r a t i o n s ) . However, i n v i v o , c o f a c t o r s and/or substrate concentration may be r a t e - l i m i t i n g ; p e n e t r a t i o n of substrate to enzyme may be r e s t r i c t e d or f a c i l i ­ t a t e d ; n a t u r a l m o d i f i e r s may be present; i n t e r a c t i o n between metabolic processes may occur and a f f e c t r e a c t i o n r a t e s . The values KJQ (a measure of enzyme-substrate a f f i n i t y ) and V (the c a p a b i l i t y of the enzyme when saturated with substrate) are v a l u a b l e , p a r t i c u l a r l y i n a comparative sense. T h e i r true relevance to a p a r t i c u l a r s i t u a t i o n i n v i v o however i s d i f f i c u l t to assess. M

A

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

X

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Another t r a p , but of our own making, must be considered. A r a p i d assay system i s very important i n enzyme p u r i f i c a t i o n and i s very convenient i n t i s s u e and species-comparisons. I t a l s o h e l p s , but i s l e s s easy to arrange, i n s t r u c t u r e - a c t i v i t y s t u d i e s . There has been a tendency i n x e n o b i o t i c enzymology f o r convenience to dominate i n the s e l e c t i o n of substates. The newcomer to the science of x e n o b i o t i c metabolism must s u r e l y be puzzled at the c e n t r a l r o l e occupied by £-nitrophenol. Unsuitable or r e s t r i c t e d s u b s t r a t e s e l e c t i o n has l e d to the p e r p e t r a t i o n of some q u i t e unwarranted g e n e r a l i s a t i o n s . In a d d i t i o n c e r t a i n convenient subs t r a t e s have proved u n s u i t a b l e i n a physico-chemical sense and t h e i r use has l e d to some very complex k i n e t i c s dominated by the p r o p e r t i e s of the s u b s t r a t e r a t h e r than those of the enzyme. Another d i f f i c u l t y rathe potential failure f approaches u s i n g separate miss an i n t e r a c t i o n betwee processes example of t h i s i s the production of a metabolite by o x i d a t i o n ( i . e . a type I process) followed by i t s conjugation (type I I p r o c e s s ) . The two r e a c t i o n s may be more than simply consecutive. S p e c i f i c examples w i l l be d i s c u s s e d below. Use

i n X e n o b i o t i c Metabolism

Studies

S u b c e l l u l a r f r a c t i o n s have been used e x t e n s i v e l y to study the d i s c r e t e steps i n x e n o b i o t i c metabolism. They are a l s o very u s e f u l i n a comparative sense. Conjugation r e a c t i o n s , l i k e other b i o t r a n s f o r m a t i o n s , may be a f f e c t e d by a number of f a c t o r s . These i n c l u d e s p e c i e s , t i s s u e , sex, s t r a i n , s t r e s s , age, time, chemicals ( i n d u c t i o n , i n h i b i t i o n , a c t i v a t i o n ) and pregnancy. The e f f e c t s of these on a p a r t i c u l a r b i o t r a n s f o r m a t i o n are conv e n i e n t l y s t u d i e d at the s u b c e l l u l a r l e v e l . For example, i f species comparisons i n v i t r o are shown to be v a l i d i n terms of i n v i v o r e s u l t s across a range of experimental animals, they can be u s e f u l l y extended to human biopsy samples, thus f u r n i s h i n g some metabolic data f o r man. The r e l a t i o n s h i p between chemical s t r u c t u r e and metabolism i s a l s o very conveniently i n v e s t i g a t e d in vitro. These v a r i o u s aspects are e x e m p l i f i e d below f o r the i n d i v i dual conjugation r e a c t i o n s . The r e a c t i o n s r e q u i r i n g a c t i v a t e d conjugand (glucuronide formation, s u l p h a t i o n , phosphorylation, a c e t y l a t i o n and methylation) are d i s c u s s e d f i r s t followed by those i n v o l v i n g a c t i v a t i o n of the x e n o b i o t i c (amino a c i d conj u g a t i o n ) . Glutathione conjugation, which depends upon the mutual i n t r i n s i c r e a c t i v i t y of both s u b s t r a t e s , i s d i s c u s s e d l a s t . Conjugation w i t h Glucuronic A c i d Mechanism and l o c a t i o n . Glucuronic a c i d conjugation i s probably the most q u a n t i t a t i v e l y important of the Type I I processes, both i n terms of p r o p o r t i o n of a p a r t i c u l a r x e n o b i o t i c being so conjugated, and i n the v a r i e t y of compounds t a k i n g p a r t

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i n the r e a c t i o n . Phenols, a l c o h o l s , c a r b o x y l i c acids and N-hydroxy compounds a l l form O-glucuronides and a number of S- and Nglucuronides have been detected. The process has r e c e n t l y been reviewed i n some d e t a i l by Dutton and co-workers (14)(15). This process i s an example of one i n which the endogenous conjugand ( g l u c u r o n i c acid) i s a c t i v a t e d to a high energy donor (uridine-5 -diphospho-a-D-glucuronic a c i d , UDPGA). The t r a n s f e r of the α-D-glucopyranuronyl group from UDPGA to the acceptor, forming £-D-glucopyranuronosides, i s c a t a l y s e d by UDPGAglucuronyl transferase (EC 2.4.1.17). The enzyme i s l o c a t e d i n the endoplasmic r e t i c u l u m of mammalian c e l l s and t h e r e f o r e appears, on s u b c e l l u l a r f r a c t i o n a t i o n , i n the microsomes. I s o l a t i o n , p r o p e r t i e s and use. The components r e q u i r e d f o r an i n v i t r o study: microsomes UDPGA x e n o b i o t i c and b u f f e r are r e a d i l y a v a i l a b l e . UDPG simple system i s p e r f e c t l metabolism. I t i s i n a p p r o p r i a t e to review assay methods i n great d e t a i l i n t h i s chapter because, i n x e n o b i o t i c metabolism, we are so o f t e n i n t e r e s t e d i n a s p e c i f i c compound or s e r i e s of compounds and the assay procedure w i l l be based on the conversion of that compound. There are, however, some recent methods that may be g e n e r a l l y u s e f u l . A method i n v o l v i n g UDP[1^C]GA u t i l i s e s an Amberlite XAD-2 column to separate unchanged UDPGA, conjugate and unchanged substrate (16). A continuous assay based on the enzymatic assay of the UDP r e l e a s e d during the t r a n s f e r has a l s o been reported (17). The i s o l a t i o n of the enzyme i s achieved by the p r e p a r a t i o n of microsomes as described above. The enzyme i s r e l a t i v e l y s t a b l e i n f r o z e n 10,000 g supernatant and i n f r o z e n microsomal p e l l e t . I f s t o r e d at -196°C ( l i q u i d n i t r o g e n ) , r a t and r a b b i t enzymes can be kept f o r many days (18).Because the enzyme i s c l o s e l y a s s o c i a t e d with the l i p o p r o t e i n microsomal membranes, f u r t h e r p u r i f i c a t i o n must be preceded by s o l u b i l i s a t i o n . A recent method (19) i n v o l v e s treatment of the microsomes with 1% Lubrol 12A9 (a condensate of dodecyl a l c o h o l with approx. 9.5 mol of ethylene oxide per mol) f o r 20 min at 4°C. The enzyme remained i n the supernatant a f t e r f u r t h e r c e n t r i f u g a t i o n i . e . s o l u b i l i s a t i o n apparently occurred. I t was then p r e c i p i t a t e d w i t h ammonium sulphate and f u r t h e r p u r i f i e d i n the presence of 0.05% L u b r o l by DEAE c e l l u l o s e chromatography. The product contained only 3 p o l y p e p t i d e s ; enzyme a c t i v i t i e s towards 2-aminophenol and 4 - n i t r o ­ phenol were increased 43- and 4 6 - f o l d r e s p e c t i v e l y . The p u r i f i e d enzyme had much improved s t a b i l i t y i n comparison with the microsomally-bound enzyme. Although there i s much k i n e t i c evidence f o r the existence of s e v e r a l glucuronyl t r a n s f e r a s e s (15), very few p a i r s of aglycone spécificités have been separated p h y s i c a l l y . One such separation i s that of the a c t i v i t i e s towards 4-nitrophenol and morphine by D e l l V i l l a r et a l (20). I t i s i n t h i s area where p u r i f i c a t i o n of the enzymes i s important. P u r i f i c a t i o n , however d e s i r a b l e i n theory and however necessary f

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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to the study of the enzyme, i s r a r e l y c a r r i e d out i n s t u d i e s of p e s t i c i d e conjugation. Our main concern i s the f a t e of the p e s t i c i d e i n the whole animal and the r o l e that the conjugation may p l a y i n the d i s p o s i t i o n of the p e s t i c i d e . Nevertheless we have to be aware of some p r a c t i c a l complications consequent on the p a r t i c u l a t e nature of t h i s enzyme. UDPGA-glucuronyl t r a n s f e r a s e i s ' l a t e n t i n f r e s h l y p r e pared microsomes. Membrane-perturbing processes such as aging, f r e e z i n g and thawing, and treatment with detergents, chaotropes, organic s o l v e n t s , a l k a l i , t r y p s i n or phospholipases may a c t i v a t e the enzyme by a f a c t o r of 40 or more (15). T h i s property, together with evidence d e r i v e d from k i n e t i c s t u d i e s , some i n v o l v i n g competitive s u b s t r a t e s , i n d i c a t e s that the enzyme i s deeply b u r i e d , p o s s i b l th inne surfac f th microsomal v e s i c l e . For t h i s reaso with T r i t o n X-100 (overal ) g x e n o b i o t i c s u b s t r a t e . This treatment should f u l l y a c t i v a t e the enzyme and remove e r r o r s due to unknown amounts of a c t i v a t i o n caused by age, storage, mechanical e f f e c t s or even an e f f e c t of the x e n o b i o t i c substrate i t s e l f . The treatment should a l s o allow access of both UDPGA and the x e n o b i o t i c to the enzyme. T h i s may be r e s t r i c t e d , p a r t i c u l a r l y i f an i o n i c substrate (e.g. a c a r b o x y l i c acid) i s being i n v e s t i g a t e d . There are two f u r t h e r problems a s s o c i a t e d with i t s use however. The amount of a c t i v a t i o n i s species-dependent. For example, the g l u c u r o n i d a t i o n of 7-hydroxychlorpromazine (21) i s a c t i v a t e d twice as much i n r a t l i v e r microsomes as i t i s i n guinea-pig l i v e r microsomes. A c t i v a t i o n i s a l s o dependent on the c o n c e n t r a t i o n of T r i t o n X100 (see Figure 3). 1

Another problem i s the d e s t r u c t i o n of UDPGA by pyrophosphorylase. This i s p a r t l y i n h i b i t e d by EDTA but a b e t t e r s o l u t i o n i s apparently the use of c i t r a t e b u f f e r which v i r t u a l l y abolishes the pyrophosphorylase a c t i o n (15). The enzyme has a pH optimum of about 7.4 though t h i s may vary somewhat with i o n i s a b l e s u b s t r a t e s . Reaction rates are l i n e a r f o r at l e a s t an hour at 37°C. Examples of use. One of the most frequent uses of the g l u c u r o n i d a t i o n system i n our l a b o r a t o r y i s i n the b i o s y n t h e s i s of glucuronides of f a e c a l m e t a b o l i t e s . These are then chromatog r a p h i c a l l y compared with suspected glucuronides found i n the u r i n e or i n the b i l e of t r e a t e d animals. Figure 4 i l l u s t r a t e s the formation of the glucuronide of a n t i - 1 2 - h y d r o x y - [ l ^ C l e n d r i n under the f o l l o w i n g c o n d i t i o n s : volume, 5 ml; hydroxyendrin, 0.004 mM; UDPGA, 0.75 mM; washed r a b b i t l i v e r microsomes (4.2 mg/ml) i n 0.1 M TRIS-HC1 b u f f e r (pH 7.4 at 37°C); i n c u b a t i o n temperature, 37°C. P o r t i o n s (1 ml) were withdrawn at i n t e r v a l s and p a r t i t i o n e d between benzene and water which were then r a d i o assayed to a f f o r d the proportions of unchanged substrate and conjugate r e s p e c t i v e l y (22). T r i t o n X-100 had no e f f e c t on t h i s r e a c t i o n , p o s s i b l y because the very l i p o p h i l i c a n t i - 1 2 -

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

0.1

0.2

0.3

0.4

0.5

Concentration of Triton X-100 (%) ure 3.

Effect of Triton X-100 on glucuronidation: ( ), rat liver microsomes; ( ), guinea pig liver microsomes.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CRAWFORD AND HUTSON

Figure 4.

Type

II

Metabolism

Time course of the glucuronidation of C anti-12-hydroxyendrin by rabbit liver microsomes 14

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192

XENOBIOTIC METABOLISM

hydroxyendrin was r e a d i l y a c c e s s i b l e to the enzyme. The common experimental animals, e.g. guinea-pig, r a t , mouse, hamster and dog, a l l possess reasonable amounts of h e p a t i c microsomal g l u c u r o n y l t r a n s f e r a s e . L i t t e r s t e t a l (23) have compared the h e p a t i c enzyme i n rhesus monkey ( c u r r e n t l y i n short supply) with species which may be used as a l t e r n a t i v e t e s t animals. A c t i v i t i e s towards 4-nitrophenol (nmol per min per mg p r o t e i n ) were: rhesus monkey, 7; s q u i r r e l monkey, 8-11 (sex d i f f e r e n c e ) ; common t r e e shrew (9-12); miniature p i g (6-9); Sprague-Dawley r a t (2-3). These measurements were made without the a d d i t i o n of detergent. Conditions were: s u b s t r a t e , 0.2 mM; UDPGA, 3.3 mM; p r o t e i n , 1 mg/ml; T r i s b u f f e r pH 7.4, 1 mM. The cat and other F e l i d a e excrete l i t t l e o r no glucuronide conjugates of x e n o b i o t i c s (24). The h i g h K value f o r 4-nitrophenol with cat l i v e r g l u c u r o n y l t r a n s f e r a s t r a n s f e r a s e (25) i s i n s i m i l a r l y do not excrete many glucuronide conjugates and have very low enzyme a c t i v i t i e s i n l i v e r . These are examples of the i n v i t r o s u b c e l l u l a r r e s u l t s comparing w e l l w i t h the s i t u a t i o n i n v i v o . The occurrence of g l u c u r o n y l t r a n s f e r a s e i n b i r d s g e n e r a l l y has not been s t u d i e d . F i s h excrete glucuronides (26) and have been shown to possess h e p a t i c g l u c u r o n y l t r a n s f e r a s e a c t i v i t y towards 2-aminophenol (27) 3 - t r i f l u o r o m e t h y l - 4 - n i t r o phenol (26), and 4-nitrophenol (28). Glucuronide formation i s important i n man but the h e p a t i c enzyme has not been examined in detail. Another important use of i n v i t r o techniques i s f o r the comparison of b i o t r a n s f o r m a t i o n i n d i f f e r e n t t i s s u e types. However, a f t e r a survey u s i n g a s e n s i t i v e me thylumb e l l i f e r o n e assay, A i t i o (29) concluded that l i v e r i s the most important organ of glucuronide s y n t h e s i s . I t was estimated that the whole g a s t r o i n t e s t i n a l t r a c t possessed only 15-20% of the enzyme a c t i v i t y found i n l i v e r . Some r e s u l t s are summarised i n Table I. Low enzyme a c t i v i t y has a l s o been found i n the p l a c e n t a of s e v e r a l s p e c i e s , i n c l u d i n g man (30). The r e l a t i v e a c t i v i t i e s found f o r v a r i o u s t i s s u e s are p a r t l y a f u n c t i o n of the choice of s u b s t r a t e . For example, b r a i n , h e a r t , f a t and diaphragm possess very low a c t i v i t i e s towards methylumbelliferone (30) but q u i t e h i g h activités towards the c a r b a r y l m e t a b o l i t e , 1-naphthol (31). This demonstrates that model s u b s t r a t e s serve only as a guide; f o r r e l e v a n t i n f o r m a t i o n on a s p e c i f i c chemical, only s t u d i e s on that chemical w i l l r e a l l y s u f f i c e . T o x i c o l o g i c a l s i g n i f i c a n c e . The value of g l u c u r o n i d a t i o n l i e s i n the dramatic change i n p o l a r i t y that the process confers. The glucuronides are also very r e a d i l y secreted i n b i l e or v i a the kidneys and thus removed from the body. G l u c u r o n i d a t i o n can a l s o prevent c e r t a i n types of metabolite from being f u r t h e r b i o a c t i v a t e d to r e a c t i v e species such as the quinones which may be formed from aromatic d i h y d r o d i o l s (32). The process i s almost always a d e t o x i f i c a t i o n , however i n some important

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

6.

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Metabolism

193

cases the reverse i s observed. The g l u c u r o n i d a t i o n o f Ν-hydroxyarylacetamides (e.g. N-hydroxyphenacetin, Figure 5) a f f o r d s chemically r e a c t i v e molecules capable of i n t e r a c t i n g with t i s s u e macromolecules (33). The demonstration of t h i s type o f b i o a c t i v a t i o n i s a valuable use of i n v i t r o t e s t systems.

Table I. Glucuronidation o f methylumbelliferone in rat tissues

Enzyme a c t i v i t y Tissue

Liver Duodenal mucosa Adrenal glands Kidneys Spleen Lungs Thymus Heart Brain

nmol product p e r g

460 260 170 150 30 28 19 1.4 0.8

nmol product

3200 96 63 120 35 34 9 1.1 1.3

The use of separated s u b c e l l u l a r f r a c t i o n s may f a i l to r e v e a l p o s s i b l e i n t e r a c t i v e e f f e c t s or f u n c t i o n a l r e l a t i o n s h i p s between the f r a c t i o n s . For example, the a d d i t i o n of UDPGA to r a t l i v e r microsomes increases t h e i r rate of 12-hydroxylation of d i e l d r i n (34). The r a t i o n a l e f o r adding UDPGA t o an o x i d a t i v e system was that sjn-12-hydroxydieldrin (Figure 6), although more h y d r o p h i l i c than d i e l d r i n , i s s t i l l a very l i p o p h i l i c molecule, p a r t i c u l a r l y i n view of the hydrogen bonding of the hydroxy1 group to the epoxide oxygen. Thus, when formed as a metabolite v i a the a c t i o n o f microsomal mon o-oxygen as e, i t would remain near i t s s i t e of formation, p o s s i b l y i n h i b i t i n g f u r t h e r h y d r o x y l a t i o n of d i e l d r i n . This e f f e c t was f i r s t noted by von Bahr and B e r t i l s s o n (35) with demethylimipramine. The f u n c t i o n a l r e l a t i o n s h i p between microsomal monooxygenase, epoxide hydratase and glucuronyl t r a n s f e r a s e has r e c e n t l y been i n v e s t i g a t e d i n l i v e r microsomes and i n i s o l a t e d hepatocytes (32). The r e s u l t s provide a good i l l u s t r a t i o n of one of the l i m i t a t i o n s of the s u b c e l l u l a r approach. In the hepatocyte and i n microsomes f o r t i f i e d with NADPH and UDPGA, naphtha­ lene i s oxygenated to i t s 1,2-oxide which i s cleaved t o a d i h y d r o d i o l (by epoxide hydratase) which, i n t u r n , i s conjugated with g l u c u r o n i c a c i d (Figure 7). However, microsomes a f f o r d the d i h y d r o d i o l as the major metabolite; hepatocytes a f f o r d the glucuronide. Only by using e i t h e r more microsomal p r o t e i n o r the

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

194

XENOBIOTIC METABOLISM

Figure 5.

Conjugation in the formation of reactive metabolites from phenacetin

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

6.

CRAWFORD AND HUTSON

Cl

Type

Cl

Figure 6.

II

195

Metabolism

Cl

Hydroxyhtion and glucuronidation of dieldrin

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

196

XENOBIOTIC

mo no-oxygenase plus 0

2

METABOLISM

epoxide hydratase + H 0 2

microsomal glucuronyl transferase + UDPGA

Figure 7.

Rehtionship between the enzymes affecting the metabolism of naphthalene

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

6.

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Type

II

Metabolism

197

a l l o s t e r i c e f f e c t o r of g l u c u r o n y l t r a n s f e r a s e , UDP-N-acetylglucosamine, could microsomes be f o r c e d to y i e l d reasonable q u a n t i t i e s of glucuronide. T h i s e f f e c t o r may a l s o c o m p e t i t i v e l y i n h i b i t the d e s t r u c t i v e e f f e c t of the pyrophosphorylase on UDPGA in vitro. Conjugation with Sulphate Mechanism and l o c a t i o n . Conjugation with sulphate i s mediated by the s u l p h o t r a n s f e r a s e enzymes ( h i s t o r i c a l l y sulphok i n a s e s ) . The donor s u b s t r a t e , which contains the a c t i v a t e d sulphate group, i s 3'-phosphoadenosine-S -phosphosulphate (PAPS). Acceptor groups are p r i n c i p a l l y p h e n o l i c but an a l c o h o l i c or primary amine f u n c t i o n can a l s o be sulphated v i a the same mechanism. I r r e s p e c t i v e of the chemical nature of the f i n a l sulphated product, sulphoconjugatio pathway: a c t i v a t i o n of sine 5 -phosphosulphate (APS) and then 3 -phosphoadenosine 5 phosphosulphate (PAPS), followed by t r a n s f e r of the sulphate group from PAPS to s u i t a b l e acceptors: 1

1

f

2

ATP

+ S0 ~

*

APS

+ ATP

*

4

PAPS + Acceptor

PAP

APS

f

+ PP£

(1)

PAPS + ADP

(2)

+ sulphated acceptor

(3)

The enzymes c a t a l y s i n g these r e a c t i o n s are (1) ATP-sulphate adenylyl t r a n s f e r a s e (ATP-sulphurylase); (2) ATP-adenylyl sulphate 3 -phosphotransferase (APS-kinase) and (3) an appropriate s u l p h o t r a n s f e r a s e (EC 2.8.2). In mammalian c e l l s the enzymes r e s p o n s i b l e f o r the p r o d u c t i o n of PAPS from sulphate are l o c a l i s e d i n the c y t o s o l . Sulphotransferases are l o c a t e d i n both the microsomal and the c y t o s o l i c f r a c t i o n s of a wide v a r i e t y of t i s s u e s ; t h e i r general t i s s u e d i s t r i b u t i o n resembles that of the UDP-glucuronyltransferases, except that they are a l s o abundant i n p l a c e n t a . Boundaries of t h e i r s p e c i f i c i t i e s are more c l e a r than those of the g l u c u r o n y l t r a n s f e r a s e s , and d i s t i n c t i v e phenol, s t e r o i d and arylamine s u l p h o t r a n s f e r a s e a c t i v i t i e s have been detected. Other t r a n s f e r a s e s deal apparently s o l e l y with endogenous compounds, e.g. cerebroside and p o l y s a c c h a r i d e sulphot r a n s f erases. Simple endogenous sulphates i n c l u d e s those of s t e r o i d s , a d r e n a l i n e , t r i - i o d o t h y r o n i n e and s e r o t o n i n . The p i c t u r e emerges ( c f . the g l u c u r o n y l t r a n s f e r a s e s ) of the sulphot r a n s f e r a s e s r e s p o n s i b l e f o r the s u l p h a t i o n of small molecules being f r e e l y s o l u b l e i n the c y t o s o l , whereas those that are i n v o l v e d i n the assembly of l a r g e r molecules are arranged, together with other r e q u i s i t e b i o s y n t h e t i c enzymes, i n assemblyl i n e f a s h i o n on the membranes of the endoplasmic r e t i c u l u m and the G o l g i apparatus. I t i s extremely d o u b t f u l whether any of these bound enzymes p l a y any r o l e i n the metabolism of xenob i o t i c s (36). f

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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XENOBIOTIC METABOLISM

I s o l a t i o n , p r o p e r t i e s and use. The u n c e r t a i n t y i n the t o t a l number, p r e c i s e r o l e s and s p e c i f i c i t i e s of the s u l p h o t r a n s f e r a s e s r e f l e c t s the f a c t that they are exceedingly d i f f i c u l t to p u r i f y and to separate from one another. Moreover, some of them aggre­ gate and/or change conformation under c e r t a i n c o n d i t i o n s ; m u l t i p l e peaks of s i m i l a r a c t i v i t y , which may o r may not be due to the same enzyme, appear on chromatography columns and are a constant hindrance to the e x p e r i m e n t a l i s t . A s u l p h o t r a n s f e r a s e has been p a r t i a l l y p u r i f i e d from bovine kidney (acetone powder) using 4-nitrophenol as the assay sub­ s t r a t e (37). The a c t i v i t y of the enzyme was measured using [35s]PAPS £ the assay mixture. Substrate s p e c i f i c i t y was i n v e s t ­ i g a t e d and i t was found to be r e l a t i v e l y s p e c i f i c f o r simple a r y l sulphate formation as shown i n Table I I n

Table I I . R e l a t i v bovine kidney s u l p h o t r a n s f e r a s e

Substrate

Relative specificity

4-Nitrophenol 4-Hydroxybenzaldehyde 4-Chlorophenol 1-Naphthol Phenol o-Cresol m-Cresol 3-Nitrophenol 3-Hydroxybenzaldehyde

100 75.9 79.7 32.5 6.5 21.7 8.3 49.8 21.0

No a c t i v i t y could be demonstrated towards e t h a n o l , propan-Ι­ οί, b u t a n - l - o l , or towards a range of s t e r o i d s . The i s o l a t i o n of N-hydroxy-2-acetylaminofluorene (N-0H-2AAF) s u l p h o t r a n s f e r a s e has r e c e n t l y been achieved from the c y t o s o l f r a c t i o n s of male and female r a t l i v e r s (the l a t t e r possess very low a c t i v i t y ) (38). A 2000-fold p u r i f i c a t i o n with a y i e l d of over 12% was achieved using the f o l l o w i n g procedure: ammonium sulphate f r a c t i o n a t i o n , DEAE-cellulose column chromatography, hydroxya p a t i t e column chromatography, sephadex G-200 g e l f i l t r a t i o n , i s o e l e c t r i c f o c u s s i n g and, f i n a l l y , more sephadex G-200 g e l f i l t r a t i o n . The f i n a l p r e p a r a t i o n was homogenous on a n a l y t i c a l d i s c g e l e l e c t r o p h o r e s i s . The p u r i f i e d enzyme had a c t i v i t y towards 4-nitrophenol with an approximately 1600-fold i n c r e a s e i n s p e c i f i c a c t i v i t y over the crude homogenate, but i t had very low a c t i v i t y towards endogenous s t e r o i d s and s e r o t o n i n . PAPS was used as the sulphate donor i n these assay mixtures and was synthesised e n z y m a t i c a l l y (39). The pure enzyme was very u n s t a b l e , e s p e c i a l l y i n d i l u t e s o l u t i o n s . T h i o l compounds were found to have a s t a b i l ­ i s i n g e f f e c t and t h i o l b l o c k i n g reagents were potent i n h i b i t o r s .

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Metabolism

199

The i n v o l v e d nature of these p u r i f i c a t i o n s suggests that the study of phase I I sulphoconjugations u s i n g p u r i f i e d sulphotrans­ ferase from v a r i o u s animal species and animal t i s s u e s i s impract­ i c a l at present. An i n v i t r o assay system f o r studying these r e a c t i o n s u s i n g crude s u b c e l l u l a r f r a c t i o n s has been developed by Mulder et a l (33). I t comprises c y t o s o l (600 μg p r o t e i n / m l ) , x e n o b i o t i c (0.5 mM), T r i s - H C l b u f f e r (100 mM, pH 8.0) and a PAPSgenerating system (PAPS-GS): 3 5 - a d e n o s i n e diphosphate (PAP, 10 μΜ) and 4-nitrophenyl sulphate (10 mM). In t h i s assay mixture 4-nitrophenyl sulphate i s used to convert PAP i n t o PAPS, a r e a c t i o n presumably c a t a l y s e d by a phenolsulphotransferase i n the c y t o s o l . The r a t e of formation of 4-nitrophenol i s spectrophotom e t r i c a l l y determined and i s c a l c u l a t e d by s u b t r a c t i n g the amount of 4-nitrophenol r e l e a s e d i n a c o n t r o l i n c u b a t i o n mixture ( x e n o b i o t i c absent) fro x e n o b i o t i c . The r a t e o c u l a t e d from the amount of 4-nitrophenol r e l e a s e d u s i n g a molar e x t i n c t i o n c o e f f i c i e n t of 17,500 M" cm~l (at pH 8.0). This assay procedure makes an important c o n t r i b u t i o n to the f i e l d of xeno­ b i o t i c metabolism i n that i t provides a cheap and simple method f o r studying the mechanisms and r a t e s of phase I I sulphate con­ j u g a t i o n r e a c t i o n s using a PAPS-GS and c y t o s o l . H i t h e r t o PAPS has been commercially a v a i l a b l e i n the l a b e l l e d form only and i t s enzymic s y n t h e s i s i s a tedious process (39)(40). A more t r a d ­ i t i o n a l assay procedure i s that described by Wu and Straub (38) where PAPS i s used at a c o n c e n t r a t i o n of approximately 0.3 mM when substrate concentration i s 0.2 mM. f

1

1

Examples of use. Harmol (Figure 8) i s a good substrate f o r phenol sulphotransferase i n r a t l i v e r 600 g f r a c t i o n (41) but harmalol (Figure 8) i s a very poor s u b s t r a t e . The reason f o r the d i f f e r e n c e i n r a t e of s u l p h a t i o n of these two substrates i s unknown, but the f i n d i n g s agree with the d i f f e r e n t r a t e s and modes of conjugation ( s u l p h a t i o n and g l u c u r o n i d a t i o n ) of the two compounds found i n v i v o . The metabolism of harmol i l l u s t r a t e s a problem that o f t e n a r i s e s when d i s c u s s i n g the phase I I conjugation of x e n o b i o t i c s with sulphate. Sulphation of a x e n o b i o t i c or i t s metabolite i s p r a c t i c a l l y always accompanied by conjugation with g l u c u r o n i c a c i d . Glucuronic a c i d conjugation o f t e n predominates and t h i s has been assumed to be due to a l i m i t e d supply of sulphate i n v i v o . Mulder and coworkers have developed a method f o r the simultan­ eous measurement of UDP-glucuronyltransferase and phenolsulphot r a n s f e r a s e from r a t l i v e r i n v i t r o , using harmol as substrate and 600 g supernatant as the enzyme source (42). T r i t o n X-100 was used to a c t i v a t e g l u c u r o n y l t r a n s f e r a s e ( S e c t i o n 5.1) and was shown to have no e f f e c t on the a c t i v i t y of phenolsulpho­ t r a n s f erase. The amount of the conjugates formed was measured f l u o r o m e t r i c a l l y and the a c t i v i t i e s of the enzymes were expres­ sed as a r b i t r a r y u n i t s of fluorescence recovered from a t i c a n a l y s i s of the incubates. At low substrate concentrations

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC

METABOLISM

glucuronidation sulphation phosphorylation (?)

,οχ COCH

3

(reactive) Figure 9.

Acetyhtion and conjugation in the activation of aromatic amines

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

6.

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Metabolism

201

( arene oxide -> d i h y d r o d i o l -> catechol monomethylcatechol. Most of the enzyme a c t i v i t y i s present i n the c y t o s o l of v a r i o u s t i s s u e s . Phenol 0-methy1transferase, a microsomal enzyme, has been detected i n v i t r o (68) but i t s s i g n i f i c a n c e i n v i v o i s unknown because the 0-methylation of monohydric phenols apparently does not occur i n v i v o . A p o t e n t i a l l y u s e f u l general assay method i n v o l v i n g the use of the t r i t i a t e d cosubstrate S-adenosy1-L[methyl-3H]methionine has been described (69). The r a d i o a c t i v e methylated product i s e x t r a c t e d i n t o organic solvent f o r r a d i o assay. S-Methylation. ^ - M e t h y l a t i o n i s a general pathway of metabo l i s m of the thiopyrimidone a n t i t h y r o i d drugs such as 6-propyl2 - t h i o u r a c i l . T e t r a h y d r o f u r f u r y l mercaptan i s a l s o S-methylated i n an S-adenosylmethionine-dependent enzymatic r e a c t i o n . The enzyme was found i n the microsomal f r a c t i o n of l i v e r , kidney and small i n t e s t i n e (70). Thiophenol l i b e r a t e d during the metabolism of dyfonate (Figure 11) i s methylated (71). This r e a c t i o n has not been s t u d i e d i n v i t r o . N-Methylation. P y r i d i n e d e r i v a t i v e s are N-methylated but the r e a c t i o n s are q u a n t i t a t i v e l y of minor importance. The most widely s t u d i e d N-methylation r e a c t i o n s are those i n v o l v i n g the b i o g e n i c amines. For example, phenylethanolamine N-methyItransferase (EC 2.1.1) (involved i n the b i o s y n t h e s i s of epinephrine) has been s t u d i e d r e c e n t l y and found to methylate non-aromatic substrates as w e l l as aromatic ones (e.g. Figure 12, R = p r o p y l , c y c l o h e x y l , c y c l o h e x - 3 - e n y l , c y c l o - o c t y l ) . The enzyme has an absolute r e q u i r e ment f o r a hydroxy1 group at the 3 - p o s i t i o n on an e t h y l s i d e chain (72). Methylation r e a c t i o n s have not featured widely i n p e s t i c i d e metabolism; however, there i s enough information d e r i v e d from endogenous biochemistry and drug metabolism to be u s e f u l should 1

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

CRAWFORD AND i i u T S O N

Type

II

Metabolism

K3

Et

EtO

Ο

OH

Me S

MeS

mercapturic acid

Pr-N^^O

Glutathione in the metabolism of thiocarbamate herbicides

and thiotriazine

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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b i o t r a n s f o r m a t i o n without being obvious i n the f i n a l products. Even with the well-known glutathione-dependent demethylation of dimethylphosphate t r i e s t e r i n s e c t i c i d e s , most of the S-methylg l u t a t h i o n e formed i s metabolised to CO2 and very l i t t l e me t h y 1 mercapturic a c i d i s excreted. C l e a r l y the use of s u b c e l l u l a r f r a c t i o n s i s i n d i c a t e d i n d i s c o v e r i e s of t h i s type. The f a t e of the v i n y l phosphate i n s e c t i c i d e , dimethylvinphos, may be used to i l l u s t r a t e t h i s p o i n t f u r t h e r . The main r a d i o a c t i v e metabolites d e r i v e d from l^C-phenyl l a b e l l i n g i n v i v o were de-methyldimethylvinphos and a metabolite derived from the phenylvinyloxy group, 1-(2,4-dichlorophenyl)ethanol (as glucuronide) (4). The demethylation, p r e d i c t a b l y , was e f f e c t e d by ( d i a l y s e d ) c y t o s o l and g l u t a t h i o n e . 2,4-Dichlorophenylethanol was assumed to be d e r i v e d from 2,4-dichloroacetophenon by the r e d u c t i v e d e c h l o r i n a t i o dimethylvinphos (2,4-dichlorophenacyl c h l o r i d e ) . The mechanism of the r e d u c t i v e d e c h l o r i n a t i o n was unknown u n t i l we s t u d i e d the metabolism of the phenacyl c h l o r i d e i n r a t l i v e r f r a c t i o n s . The f o l l o w i n g sequence of reactions was d i s c o v e r e d u s i n g c y t o s o l : ( i ) spontaneous r e a c t i o n of phenacyl h a l i d e with g l u t a t h i o n e to a f f o r d 2 , 4 - d i c h l o r o p h e n a c y l - g l u t a t h i o n e , ( i i ) enzyme-catalysed r e a c t i o n of the l a t t e r with another molecule of g l u t a t h i o n e to form o x i d i s e d g l u t a t h i o n e and the phenacyl anion which rearranged to 2,4-dichloroacetophenone (133). Microsomes and NADPH simply reduced the keto group of 2,4-dichlorophenacyl c h l o r i d e to give the c h l o r o h y d r i n (not observed i n the i n v i v o metabolism) . Thus g l u t a t h i o n e enters the metabolic pathways of dimethylvinphos at three p o i n t s (Figure 18) but i t i s s c a r c e l y observed i n the excreted m e t a b o l i t e s . In t h i s s e r i e s of experiments we a l s o used the i n v i t r o technique to demonstrate that the a d m i n i s t r a t i o n of phénobarbital to r a t s (which p r o t e c t e d them 1 0 - f o l d against the acute o r a l t o x i c i t y of dimethylvinphos) induced the a c t i v i t y of the c y t o s o l demethylating enzyme 2 - f o l d . Dimethylvinphos e x h i b i t s a large d i f f e r e n c e i n acute t o x i c i t y to r a t and dog ( r a t > dog); dog l i v e r c y t o s o l was shown to demethylate dimethylvinphos at about twice the rate of r a t l i v e r c y t o s o l (4). Thus, the enzyme could have some r o l e i n the s e l e c t i v e t o x i c i t y of dimethylvinphos. This example i l l u s t r a t e s s e v e r a l important f e a t u r e s : d i r e c t g l u t a t h i o n e conjugation of parent molecule ( e f f e c t i n g detoxification) ( i i ) g l u t a t h i o n e conjugation a f t e r b i o a c t i v a t i o n by primary metabolism ( h y d r o l y s i s i n t h i s case) ( i i i ) g l u t a t h i o n e conjugation by attack on an e l e c t r o p h i l i c atom other than carbon (sulphur i n t h i s case) ( i v ) only t r a c e s of the glutathione-conjugated products (mercapturic a c i d s ) appearing i n the excreted metabolites (v) use of s u b c e l l u l a r f r a c t i o n s to i n v e s t i g a t e a species d i f f e r e n c e ( r a t versus dog) (i)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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GSH

CI Figure 18.

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GSH

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Glutathione in the metabolism of dimethylvinphos

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( v i ) use of s u b c e l l u l a r f r a c t i o n s to i n v e s t i g a t e the of drug-metabolising enzymes of an animal.

alteration

Studies of the l i v e r enzyme have shown that g l u t a t h i o n e t r a n s f e r a s e s occur i n many other animals i n c l u d i n g r a b b i t (118), p i g (118), (23), monkey (23)(134)(135), t r e e shrew (23), sheep (136), guinea-pig (122), horse (122), cow (122), mouse (122), chicken (126), and man (137). Where d e t a i l e d s t u d i e s have been c a r r i e d out (135)(136) (137) the enzymes, i n c l u d i n g those of man, have been shown to be very s i m i l a r i n p h y s i c a l p r o p e r t i e s . L i v e r i s the r i c h e s t source of the enzymes. For example, a recent study of Japanese monkey (Macaca fuscata) (134) was t y p i c a l i n showing the f o l l o w i n g a r y l t r a n s f e r a s e ( 1 , 2 - d i c h l o r o 4-nitrobenzene) a c t i v i t i e s (μιιιοΐ/min/mg p r o t e i n ) : l i v e r , 18; spleen, 2.6; kidney, 2.1 p l a c e n t a , 0.3; pancreas has r e c e n t l y been reported i n small i n t e s t i n e s (138)(139) and i n leucocytes (140). Enzyme measurements have a l s o been used to assess glutathione conjugation during development of the neonate. A l k y l t r a n s f e r a s e a c t i v i t y i n the r a t neonate i s very low f o r about 6 days and then r i s e s s t e a d i l y f o r about 40 days to the adult l e v e l (141). Trans­ f e r a s e Β (measured as a r y l t r a n s f e r a s e ) i s present at b i r t h at about one f i f t h of the adult l e v e l and r i s e s s t e a d i l y f o r about 40 days (142). However, i n a l l general statements about the presence of g l u t a t h i o n e t r a n s f e r a s e i n various s p e c i e s , t i s s u e s or a l t e r e d s t a t e s , the s u b s t r a t e used f o r assay must be noted before the relevance to one's own work i s assessed. The importance of g l u t a t h i o n e conjugation i n l i m i t i n g the c y t o t o x i c , mutagenic and c a r c i n o g e n i c a c t i o n of e l e c t r o p h i l i c compounds (143) i s gener­ a t i n g much research i n t o the species and t i s s u e d i s t r i b u t i o n of the enzyme(s). However, too much r e l i a n c e on r e s u l t s d e r i v e d from t e s t s using substrates (e.g. p o l y c y c l i c aromatic hydrocarbon epoxides) unrelated to one's own problem may w e l l prove to be only of l i m i t e d v a l u e . T o x i c o l o g i c a l s i g n i f i c a n c e . Glutathione conjugation r e s u l t s i n a dramatic change i n the p h y s i c a l p r o p e r t i e s of a molecule, u s u a l l y l e a d i n g to a l o s s of b i o a c t i v i t y . The conjugate i s i d e a l l y s t r u c t u r e d f o r b i l i a r y s e c r e t i o n and t h e r e f o r e i t i s e f f i c i e n t l y removed from the l i v e r . Other enzymes e f f i c i e n t l y convert the conjugate i n t o a mercapturic a c i d that i s r e a d i l y excreted v i a the u r i n e . However, perhaps the most important f u n c t i o n of t h i s conjugation process i s the p r o t e c t i o n i t a f f o r d s against e l e c t r o p h i l i c compounds, be they ingested as such or generated w i t h i n the organism v i a metabolism. Without t h i s p r o t e c t i o n mammals would be much more s u s c e p t i b l e than they are to low doses of teratogens, mutagens, carcinogens and c y t o t o x i c compounds.

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Other Uses of S u b c e l l u l a r F r a c t i o n s i n X e n o b i o t i c Metabolism Studies A r e l a t i v e l y recent use of s u b c e l l u l a r f r a c t i o n s i s i n b a c t e r i a l t e s t systems f o r mutagenicity of the type developed by Ames and coworkers (144). The f r a c t i o n commonly used i s a 9000 g f r a c t i o n (S-9 f r a c t i o n ) from the l i v e r s of r a t s t r e a t e d with an A r a c h l o r (to induce microsomal enzymes). I t s r o l e i n the t e s t system i s the p r o v i s i o n of a mammalian metabolism c a p a b i l i t y f o r the ( p o s s i b l e ) a c t i v a t i o n of i n t r i n s i c a l l y i n a c t i v e compounds. This may not commonly be seen as a use i n metabolism s t u d i e s ; but i f the b a c t e r i a are regarded as a bioassay technique f o r the d e t e c t i o n of mutagens, the system i s a u s e f u l a d d i t i o n to the techniques used f o r the study of metabolism i n v i t r o The main component of the b i o a c t i v a t i o microsomal mono-oxygenas a more 'potent S-9 f r a c t i o n ) . However, i t w i l l be c l e a r from the v a r i o u s r e a c t i o n s discussed above that some of the type I I r e a c t i o n s e f f e c t the b i o a c t i v a t i o n of c e r t a i n m e t a b o l i t e s . They a l s o e f f e c t the d e a c t i v a t i o n of many compounds and t h e i r metabo l i t e s . The p o t e n t i a l l y great p r e d i c t i v e value of these t e s t systems has l e d to t h e i r widespread, but o f t e n u n c r i t i c a l use. The importance of a standardised p r e p a r a t i o n c o n t a i n i n g a c t i v e microsomal mono-oxygenase i s appreciated but the r o l e of the many other enzymes i n the S-9 f r a c t i o n has been l a r g e l y ignored. 1

The widespread use and e n t h u s i a s t i c r e c e p t i o n gained by t h i s simple, quick and p o t e n t i a l l y very u s e f u l system has l e d to a r e a c t i o n from c e r t a i n quarters and a heated debate i s c u r r e n t l y being conducted. There i s c l e a r l y a need to d e f i n e the r e a c t i o n s o c c u r r i n g i n the t e s t system. The balance between a c t i v a t i o n and d e a c t i v a t i o n i s c r i t i c a l to i t s relevance to the i n v i v o s i t u a t i o n . The s t a t e of the v a r i o u s enzymes i n the S-9 f r a c t i o n and the concentrations of the various c o f a c t o r s (many of which are described above) r e q u i r e s measurement and c o n t r o l . Species v a r i a t i o n s are important. For example i t i s p o s s i b l e that 2-aminoanthracene (mutagenic i n the presence of S-9 from r a t l i v e r ) would not give a p o s i t i v e response i f dog l i v e r S-9 f r a c t i o n s were used. The f i r s t step i n i t s b i o a c t i v a t i o n ( N - a c e t y l a t i o n , see Figures 9 and 10) i s i n o p e r a t i v e . Glutathione-dependent d e a c t i v a t i o n i s l a r g e l y i n o p e r a t i v e i n standard S-9 f r a c t i o n because, although the g l u t a t h i o n e S-transferases are present, g l u t a t h i o n e i t s e l f i s l a r g e l y destroyed by catabolism, d i l u t i o n and o x i d a t i o n (145). A study of the various enzyme a c t i v i t i e s and c o f a c t o r concentrations i n human l i v e r f r a c t i o n (prepared as S-9) would a l s o prove very u s e f u l i n the i n t e r p r e t a t i o n of r e s u l t s of the Ames t e s t .

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James, M. O., Smith, R. L. and Williams, R. T. Xenobiotica (1972), 2, 499. Dixon, P. A. F., Uwaifo, Α., Caldwell, J. and Smith, R. L. Biochem. Soc. Trans. (1974), 2, 879. Dixon, P. A. F., Caldwell, J. and Smith, R. L. Xenobiotica (1977), 7, 695. Dixon, P. A. F., Caldwell, J. and Smith, R. L. Xenobiotica (1977), 7, 707. Dixon, P. A. F., Caldwell, J. and Smith, R. L. Xenobiotica (1977 ), 7, 717. Hughey, R. P., Rankin, Β. Β., Elce, J. S. and Curthoys, N. P. Arch. Biochem. Biophys. (1978), 186, 211. Suga, T., Kumaoka, H. and Akagi, M. J. Biochem. (Tokyo) (1966), 60, 133 Chasseaud, L. F Conference of the Germa Society o Biologica Chemistry, Tübingen, 1973", Eds. L. Flohé, H. Ch. Benöhr, H. Sies, H. D. Waller and A. Wendel. G. Thieme, Stuttgart 1974, p. 90. Chasseaud, L. F. Drug Metab. Rev. (1974), 2, 185. Hutson, D. H. ACS Symposium Series, No. 29 (1976), 103. Habig, W. H., Pabst, M. J. and Jakoby, W. B. J. Biol. Chem. (1974), 249, 7130. Jakoby, W. B. Adv. Enzymol. (1978), 46, 383. Ketley, J. Ν., Habig, W. H. and Jakoby, W. B. J. Biol. Chem. (1975), 250, 8670. Jakoby, W. Β., Habig, W. H., Keen, J. Η., Ketley, J. N. and Pabst, M. J. in "Glutathione: Metabolism and Function", Eds. I. M. Arias and W. B. Jakoby, Raven Press, New York, 1976, p. 189. Hutson, D. Η., Pickering, B. A. and Donninger, C. Biochem. J. (1972), 127, 285. Usui, K., Shishido, T. and Fukami, J. Agric. Biol. Chem. (1977), 41, 2491. Hutson, D. H. Chem. Biol. Interact. (1977), 16, 315. Lawrence, R. Α., Baker, P. R. and Cuschieri, A. Agenda Papers of 575th Meeting of the Biochemical Society, Glasgow, 1978, p. 10. Grahnen, A. and Sjoholm, I. Eur. J. Biochem. (1977), 80, 573. Clarke, A. G., Cropp, P. L . , Smith, J. N . , Speir, T. W. and Tan, B. J. Pest. Biochem. Physiol. (1976), 6, 126. Hayakawa, T., LeMahieu, R. A. and Udenfriend, S. Arch. Biochem. Biophys. (1974), 162, 223. Buckpitt, A. R., Rollins, D. E., Nelson, S. D., Franklin, R. B. and Mitchell, J. R. Analyt. Biochem. (1977), 83, 168. Bedford, C. T., Crawford, M. J. and Hutson, D. H. Chemosphere (1975), 4, 311.

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125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137.

138. 139. 140. 141. 142. 143. 144. 145.

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229

Hubell, J. P. and Casida, J. E. J. Agric. Fd. Chem. (1977), 25, 404. Akhtar, M. H. and Foster, T. S. J. Agric. Fd. Chem. (1977), 25, 1017. Shishido, T., Usui, Κ., Sato, M. and Fukami, J. Pest. Biochem. Physiol. (1972), 2, 51. Kraus, P. Arch. Pharmacol. (1976), 296, 67. Lamoureux, G. L. and Davison, K. L. Pest. Biochem. Physiol. (1975), 5, 497. Habig, W. Η., Keen, J. H. and Jakoby, W. B. Biochem. Biophys. Res. Comm. (1975), 64, 501. Ohkawa, H., Ohkawa, R., Yamamoto, I. and Casida, J. E. Pest. Biochem. Physiol. (1972), 2, 95. Crayford, J. V and Hutson D H Pest Biochem Physiol (1972), 2, 295. Hutson, D. Η., Holmes Chemosphere (1976), 5, 79. Asaoka, K., Ito, H. and Takahashi, K. J. J. Biochem. (Tokyo) (1977), 82, 973. Asaoka, K. and Takahashi,K.J.J. Biochem. (Tokyo) (1977), 82, 1313. Hayakawa, T., Udenfriend, S., Yagi, H. and Jerina, D. M. Arch. Biochem. Biophys. (1975), 170, 438. Habig, W. Η., Kamisaka, Κ., Ketley, J. N . , Pabst, M. J., Arias, I.M. and Jakoby, W. B. in "Glutathione: Metabolism and Function", Eds. I. M. Arias and W. B. Jakoby, Raven Press, New York, 1976, p. 225. Clifton, G. and Kaplowitz, N. Cancer Res. (1977), 37, 788. Pinkus, L. Μ., Ketley, J. N. and Jakoby, W. B. Biochem. Pharmacol. (1977), 26, 2359. Kaplowitz, Ν., Spina, C., Graham, M. and Kuhlenkamp, J. Biochem. J. (1978), 169, 465. Baines, P. J., Bray, H. G. and James, S. P. Xenobiotica (1977), 7, 653. Hales, B. F. and Neims, A. H. Biochem. J. (1976), 160, 231. DePierre, J . W. and Ernster, L. Biochim. Biophys. Acta (1978), 473, 149. Ames, Β. Ν., McCann, J. and Yamasaki, E. Mutat. Res. (1975), 31, 347. Dean, B. J., Hutson, D. H. and Wright, A. S. Joint meeting of the American Society of Experimental Therapeutics and the Society of Toxicology, Houston, Texas, August, 1978.

RECEIVED

December

20, 1978.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

7 Methods for the Study of Metabolism of Xenobiotics i n Insect Cell and Organ Cultures EDWINP.MARKS Metabolism and Radiation Research Laboratory, Agricultural Research, Science and Education Administration,U.S.Department of Agriculture, Fargo,ND58102 MALCOLM J. THOMPSON and WILLIAME.ROBBINS Insect Physiology Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Beltsville,MD20705 Although insect organ and cell culture methodologies have been used for severa endocrinology and developmen made of these techniques in studying the metabolism of xenobiotics in insect tissues. However, as our knowledge of insect biochemistry and the necessity for environmentally compatible pesticides increase, the use of such methodologies will grow. The physiological studies that have been done with insect organ cultures so far have shown us that when experiments are carried out using appropriate techniques and carefully defined conditions, cultured tissues can be induced to behave much the same way as they do in whole insects (1). To date, studies have included such diverse processes as the biosynthesis and secretion of hormones (4,5), the production and deposition of cuticle (6,7), the biosynthesis of yolk materials (8,9), the regeneration of nerve tissues (10), and the differentiation of epidermal cells into setae (11). In all these studies, acceptable approximations of what is known to occur in vivo have also been demonstrated in vitro. Thus, it seems reasonable that cell and organ culture systems could be used to advantage for studying the metabolism of xenobiotics in insects, particularly as i t relates to mode of action. The questions, then, are what can be gained from the use of tissue culture methodology and whether the advantages justify the additional effort and expense. Organ Culture Systems Most of the physiological and biochemical studies on insect cell and organ cultures to date have been done with organ culture systems. Organ culture differs from other in vitro techniques in that an organ must be cultured for more than 24 hours under aseptic conditions and in medium that contains an adequate energy source. In such a culture, the This chapter not subject to U.S. copyright. Published 1979 American Chemical Society

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a r c h i t e c t u r e of the organ remains i n t a c t , and some degree of organotypic a c t i v i t y can be a n t i c i p a t e d (12). Under these c o n d i t i o n s the t i s s u e s a r e o f t e n v i a b l e f o r s e v e r a l weeks or even months. For example, processes such as nerve regeneration or c u t i c l e d e p o s i t i o n may begin weeks a f t e r the t i s s u e i s explanted and may continue f o r as long as 90 days (10,11). In organ c u l t u r e s the problems encountered w i t h short-term i n c u b a t i o n s , such as s u r g i c a l trauma and m i c r o b i a l contamin­ a t i o n , a r e no longer a f a c t o r i n i n t e r p r e t i n g the r e s u l t s . Advantages of Organ Culture Techniques One of the main advantages of organ c u l t u r e i s the experimental f l e x i b i l i t y . The i n v e s t i g a t o r can add or remove t i s s u e s , s u b s t r a t e s , and products a t any time during the i n c u b a t i o n p e r i o d ; can s u b s t r a t e s ; and can i n c l u d the same c u l t u r e . A l s o , the medium can be sampled at i n t e r v a l s , and substrate l e v e l s can be c o n t r o l l e d and by-products removed without s e r i o u s l y d i s t u r b i n g the system. This experimental f l e x i b i l i t y i s demonstrated by the use of a hanging drop p r e p a r a t i o n to show that the r e l e a s e of peptide neurohormones synthesized by c u l t u r e d b r a i n s could be induced by adding c u l t u r e d c a r d i a c a , which, i n turn, sequestered and stored the neurohormones r e l e a s e d i n t o the medium (13). A second advantage of the organ c u l t u r e methodology i s that a l a r g e r q u a n t i t y of metabolites can be obtained from a given amount of t i s s u e because of the prolonged i n c u b a t i o n . A l s o , feedback i n h i b i t i o n can be prevented by frequent renewal of the medium. I s o l a t i o n and p u r i f i c a t i o n of metabolites are f r e q u e n t l y e a s i e r from the r e l a t i v e l y c l e a n medium than from whole body e x t r a c t s . These advantages have been discussed p r e v i o u s l y (14). A d d i t i o n a l advantages i n c l u d e the e x c l u s i o n of unwanted t i s s u e from the c u l t u r e s and the p r e s e r v a t i o n of i n t a c t c e l l membranes i n the l i v i n g c e l l s . Thus, when one i s working w i t h whole animals, i t has been v i r t u a l l y impossible to determine whether or not α-ecdysone i n i t s e l f has any a c t i v i t y s i n c e i t i s r e a d i l y converted by adjacent t i s s u e s to 20hydroxyecdysone i n q u a n t i t i e s s u f f i c i e n t to produce an endocrine response (15). With homogenates, although t i s s u e s p e c i f i c i t y i s maintained, the c e l l membranes a r e destroyed and endogenous i n h i b i t o r s and other i n t r a c e l l u l a r products that a r e normally r e t a i n e d by the c e l l s are r e l e a s e d , and these i n t u r n induce a v a r i e t y of u n c o n t r o l l e d r e a c t i o n s . Thus, organ c u l t u r e s , i n which the i n t e g r i t y of the c e l l s i s maintained, provide a biochemical s p e c i f i c i t y that i s d i f f i c u l t to o b t a i n i n other kinds of i n v i t r o systems. F i n a l l y , a most important advantage of the organ c u l t u r e approach i s that the i n v e s t i g a t o r can i n c l u d e both the m e t a b o l i z i n g and the t a r g e t t i s s u e s i n the same system. This

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permits study of the i n t e r r e l a t i o n s h i p or i n t e r a c t i o n between the metabolism of the t e s t compound and the mode or s i t e of a c t i o n . T h i s i s i l l u s t r a t e d by our work on the metabolism of 22,25-dideoxyecdysone (16,17). Disadvantages of Organ C u l t u r e Techniques The disadvantages of organ c u l t u r e are as numerous and as apparent as the advantages. For example, i s o l a t e d t i s s u e s may l a c k substrates or c o f a c t o r s normally present i n the i n t a c t animal, which may, i n t u r n , cause minor pathways to assume a dominant r o l e , and thereby confuse the true p i c t u r e (18). Thus, i n v i t r o r e s u l t s must be compared c a r e f u l l y with what i s known from i n v i v o s t u d i e s . Some t i s s u e s do not s u r v i v w e l l i v i t r d thu cannot be used s u c c e s s f u l l example mature muscle f r e q u e n t l y y explantation Another d i f f i c u l t y l i e s i n o b t a i n i n g uncontaminated t i s s u e s f o r c u l t u r e . With holometabolous pupae or eggs, t h i s i s not a problem (19) but i n some other stages of the l i f e c y c l e , i t may be extremely d i f f i c u l t (20). The problem of contaminated t i s s u e a l s o puts a p r a c t i c a l l i m i t on the number of expiants that can be placed i n a s i n g l e c u l t u r e . For example, i f one expiant out of four i s contaminated, 75% success may be expected i n c u l t u r e s c o n t a i n i n g one expiant per c u l t u r e , l e s s than 45% success would be expected i n c u l t u r e s c o n t a i n i n g 3 expiants and there i s only a 17% chance of success i n c u l t u r e s c o n t a i n i n g s i x expiants and contamination i s a v i r t u a l c e r t a i n t y . Thus, when l a r g e amounts of t i s s u e are needed, the expiants must be c u l t u r e d s e p a r a t e l y or i n small groups, an arduous task. Even with l a r g e numbers of c u l t u r e s , the amounts of metabolites are o f t e n v a n i s h i n g l y s m a l l , so microchemical methods must be used (4). F i n a l l y , organ c u l t u r e i s expensive, both i n time and m a t e r i a l s , and should be used only when l e s s l a b o r i o u s methods are not a v a i l a b l e . Metabolism of 22,25-Dideoxyeedysone ( T r i o l ) i n Organ C u l t u r e Having discussed the use of organ c u l t u r e i n the a b s t r a c t , we would now l i k e to d i s c u s s i n some d e t a i l a study of the metabolism of the e c d y s t e r o i d 22,25-dideoxyecdysone ( t r i o l , F i g . 1 ) . In d i f f e r e n t species of i n s e c t s , or at d i f f e r e n t times or stages d u r i n g the development of the same species of i n s e c t , t h i s i n t e r e s t i n g e c d y s t e r o i d may e i t h e r enter the major pathway(s) of molting hormone metabolism and serve as a p r e c u r s o r f o r the b i o s y n t h e s i s of the i n s e c t molting hormones (21,22) or i t may be t r e a t e d as a x e n o b i o t i c and metabolized to a complex mixture of hydroxylated s t e r o i d s (22,23). The same d u a l r o l e i s r e f l e c t e d i n the b i o l o g i c a l or p h y s i o l o g i c a l a c t i v i t y and the a c t i o n of 22,25-dideoxyecdysone: In c e r t a i n i n s e c t s or b i o l o g i c a l t e s t systems, i t has molting hormone

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Ο

22,25-Dideoxyecdysone (Triol)

Figure 2.

Schematic of leg regenerate—Rose chamber technique.

The mesothoracic legs are removed from freshly molted, late instar cockroach nymphs. After 28 days the coxal stump is removed and the leg regenerate is dissected out. The test tissues are placed under the dialysis strip and the chamber is assembled and filled with medium. The test compound(s) are placed under the dialysis strip with a microsyringe.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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a c t i v i t y (21,24,25); i n a number of other i n s e c t species i t i s a potent i n v i v o i n h i b i t o r of development, metamorphosis, and r e p r o d u c t i o n (24,25,26). Our i n i t i a l s t u d i e s w i t h the t r i o l were c a r r i e d out i n Rose multipurpose t i s s u e chambers. These chambers, which we have found to be extremely u s e f u l f o r many kinds of experiments, c o n s i s t of a s i l i c o n e gasket that i s h e l d between two c o v e r s l i p s by a p a i r of metal p l a t e s . With a s t r i p of d i a l y s i s membrane to hold the l e g regenerate t i s s u e i n p l a c e , ( F i g . 2) the Rose chamber permits experimental f l e x i b i l i t y combined w i t h a high q u a l i t y o p t i c a l image f o r v i s u a l e v a l u a t i o n of the t i s s u e w i t h a compound microscope (27). A l s o , the s o f t s i l i c o n e gaskets permit the use of a m i c r o s y r i n g e to p l a c e the experimental compound i n d i r e c t contact w i t h the t i s s u e In chambers c o n t a i n i n g a t h r e s h o l ecdysone (0.05 yg/ml), c u t i c l l e g regenerates between 10 and 14 days a f t e r the molting hormone i s added (10). When the t r i o l was t e s t e d against c u l t u r e d l e g regenerates of the cockroach Leucophaea maderae ( F . ) , i t produced c u t i c l e formation i n only 27% of the regenerates and gave e r r a t i c r e s u l t s even at high doses (28) (Table I ) . The experimental r e s u l t s obtained were not r e a d i l y understood, e s p e c i a l l y s i n c e the responses that d i d occur were not c o n c e n t r a t i o n dependent. Only when the t r i o l was looked upon as a s u b s t r a t e that could be converted to b i o l o g i c a l l y a c t i v e e c d y s t e r o i d s by p e r i p h e r a l t i s s u e s such as blood or f a t body, d i d the r e s u l t s become meaningful. When we t e s t e d t h i s hypothesis by c o - c u l t u r i n g f a t body and l e g regenerates, the i n c i d e n c e of Table I.

I n d u c t i o n of c u t i c l e formation i n cockroach l e g regenerates by c e r t a i n e c d y s t e r o i d s when tested i n the presence or absence of f a t body.

Cuticle Ecdysteroids

Control 20-Hydroxyecdysone a-Ecdysone 22,25-Dideoxyecdysone 22-Isoecdysone

a

Leg Regenerates

Formation Leg Regenerates + Fatbody

Ν

%

Ν

10 14 17 22 12

10 93 82 27 8

10

0

-

-

22 10

93 0

A l l e c d y s t e r o i d s t e s t e d at 10

\ig

per chamber.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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238

To confirm these f i n d i n g s , we made a second s e r i e s of experiments i n which we s e p a r a t e ^ c u l t u r e d cockroach f a t bodies and l e g regenerates with C-labeled t r i o l f o r 6 days i n 1 ml of M20S medium i n c l e a n , s t e r i l e g l a s s s c i n t i l l a t i o n vials. Two volumes of methanol were added, and the c u l t u r e s were worked up and then analyzed f o r r a d i o l a b e l e d s t e r o i d s by t h i n - l a y e r radiochromatography. The c u l t u r e s c o n t a i n i n g only l e g regenerate t i s s u e and t r i o l produced no a d d i t i o n a l s t e r o i d a l compounds. They contained only the unmetabolized triol. However, the f a t body c u l t u r e s showed chromatographic peaks f o r t e t r a o l s , pentaols, and conjugates as w e l l as the t r i o l ( F i g . 3 ) , evidence that the metabolism of the t r i o l had proceeded v i a h y d r o x y l a t i o n and conjugation (17). Thus, our i n i t i a l hypotheses were confirmed: the t r i o l a c t s as a precursor r a t h e r than a regenerate and f a t bod capabilities. Table I I .

Compound

E f f e c t of c e r t a i n a z a s t e r o i d s and n o n s t e r o i d a l amines and an amide i n i n h i b i t i n g the a c t i v i t y of 22,25-dideoxyecdysone ( t r i o l ) i n cockroach l e g regenerate-fat body c u l t u r e s .

a

Control I II III IV V VI

All

C u t i c l e Formation

Inhibition

%

%

93 0 16 50 50 70 80

0 100 83 46 46 25 14

coumpounds t e s t e d a t 10 \xg per chamber.

From t h i s p o i n t , the work proceeded i n two d i r e c t i o n s . One t h r u s t was concerned w i t h a group of compounds c o n s i s t i n g of c e r t a i n 25-azasteroids and n o n s t e r o i d a l amines and amides ( F i g . 4) that d i s r u p t s t e r o i d metabolism and molting and metamorphosis i n i n s e c t s (29,30); these were t e s t e d f o r i n h i b i t i o n of the a c t i v i t y of the t r i o l i n inducing c u t i c l e d e p o s i t i o n i n cockroach l e g regenerate-fat body c u l t u r e s (16). The r e s u l t s obtained with s e v e r a l of these compounds i n d i c a t e d s i g n i f i c a n t i n h i b i t i o n of metabolism of the t r i o l to an a c t i v e e c d y s t e r o i d by the c u l t u r e d t i s s u e (Table I I ) . Subsequent metabolic s t u d i e s with three of the more a c t i v e

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

7.

MARKS E T A L .

Insect Cell and Organ

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TRIOL

DISTANCE

F R O M ORIGIN (CM)

Figure 3. Radiochromatograms of 4-C -ecdysteroids extracted from the medium of cockroach tissue cultures incubated with 4- C-22,25-dideoxyecdysone (triol): (A) from cultures containing leg regenerates; (B) from cultures containing leg regenerates plus fat body. 14

14

Figure 4.

Azasteroids, nonsteroidal amines, and an amide

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i n h i b i t o r s (17) demonstrated that they have a pronounced e f f e c t on the metabolism of the t r i o l i n cockroach f a t body c u l t u r e s (Table I I I ) . Taken together, these two s t u d i e s provided the f i r s t i n v i t r o p h y s i o l o g i c a l data and f i r s t biochemical evidence that c e r t a i n of the i n h i b i t o r y a z a s t e r o i d s and n o n s t e r o i d a l amines represent a new c l a s s of i n s e c t hormonal chemicals with a novel mode of a c t i o n — t h e y i n t e r f e r e with the metabolism of the endogenous molting hormones of insects. Table I I I .

Inhibitory Compounds

The e f f e c t s o f an a z a s t e r o i d açji n o n s t e r o i d a l amines on the metabolism of 4- C-22,25-dideoxyecdysone ( t r i o l ) i n cockroach f a t body c u l t u r e s during an i n c u b a t i o n p e r i o d of 6 days

% Unmetabolized ^ 22,25-Dideoxyecdysone

% Metabolites

Conjugates Pentaols T e t r a o l s

Control II III IV

45.5 6.0 15.9 18.7

35.6 55.0 40.8 34.7

11.3 0.0 5.6 5.2

7.6 39.0 37.8 41.5

The concentrations o f the i n h i b i t o r y compounds and of 422,25-dideoxyecdysone were 10 yg each per c u l t u r e f l a s k .

C-

^ Determined by r a d i o TLC analyses.

The second t h r u s t , which i s perhaps more germane to the metabolism of x e n o b i o t i c s , i n v o l v e d over 100 c u l t u r e s of f a t body that were incubated with t r i o l . The p r i n c i p a l pathways of metabolism of t r i o l i n the f a t body c u l t u r e s turned out to be h y d r o x y l a t i o n and conjugation, as they are i n v i v o . I n t e r e s t i n g l y , enzymic h y d r o l y s i s of the conjugate f r a c t i o n showed no unmetabolized t r i o l . The major t e t r a o l metabolite was 22-deoxyecdysone, and other t e t r a o l and pentaol metabolites were hydroxylated a t the 25, 26, or 20 p o s i t i o n s ( 1 7 ) ( F i g . 5 ) . Most of the metabolites found had been p r e v i o u s l y i s o l a t e d from the f r a s s of t r i o l - f e d l a r v a e of the tobacco hornworm Manduca sexta (L.) (23). I n t h i s r e s p e c t , the metabolic pathways f o r t r i o l i n c u l t u r e d cockroach f a t body resembled the pathways that occur i n hornworm l a r v a e . A l s o , i n both cases the mechanism f o r h y d r o x y l a t i o n of the t r i o l a t C-22 appears to be l a c k i n g . When samples of the t e t r a o l and p e n t a o l f r a c t i o n s were t e s t e d against c u l t u r e d l e g regenerates,

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TETRADS

PENTAOLS OH

Η

OH

-Ι­

OH

22,25-DFDEOXYECDYSONE (TRIOL)

OH

O H

Figure 5. Hydroxylation of 22,25-dideoxyecdysone (triol) by cockroach fat body cultures

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both f r a c t i o n s induced c u t i c l e formation (Table I V ) . Since the l e g regenerates cannot m e t a b o l i c a l l y a c t i v a t e the t r i o l d i r e c t l y , they are probably unable to e f f i c i e n t l y hydroxylate the C-25 p o s i t i o n . In a d d i t i o n , assuming that 22-deoxyecdysone, l i k e α-ecdysone, i s e f f e c t i v e i n the l e g regenerate system only a f t e r metabolic conversion (31), then f u r t h e r metabolism of the t e t r a o l and pentaol metabolites by the l e g regenerates may a l s o occur. Thus, both f a t body and l e g regenerate t i s s u e may be necessary to t r a v e r s e the e n t i r e pathway from t r i o l to an e c d y s t e r o i d ( s ) with molting hormone a c t i v i t y i n the l e g regenerate system. Table IV.

E f f e c t of metabolite f r a c t i o n s i s o l a t e d from cockroach f a t bod t i s s u c u l t u r e incubated with 22,25-dideoxyecdyson by c u l t u r e d g regenerates

C u t i c l e Formation

Metabolite Fractions

Ν

Control Tetraol Pentaol

4 5 4

% 0 62 80

X days

10.8 11.3

Tested at 10 yg e q u i v a l e n t s as determined

by UV analyses.

This study i s s t i l l i n progress, and a number of questions remain unanswered. One of these concerns the b i o l o g i c a l a c t i v i t y of the v a r i o u s t e t r a o l and pentaol metabolites of the t r i o l that are produced i n f a t body c u l t u r e s . Other questions i n v o l v e the s i t e and mode of a c t i o n of c e r t a i n of the i n h i b i t o r y a z a s t e r o i d s and n o n s t e r o i d a l amines. During the course of t h i s work, we learned c e r t a i n things that may be of use to i n v e s t i g a t o r s who use s i m i l a r techniques: One of these i s that the use of a n t i b i o t i c s to c o n t r o l contamination must be approached with extreme c a u t i o n . We found that the presence of gentamicin, an a n t i b i o t i c commonly used i n t i s s u e c u l t u r e s , i n t e r f e r e d with the metabolism of the t r i o l i n f a t body c u l t u r e s . I t was a l s o observed that the amount of s u b s t r a t e and/or the length of i n c u b a t i o n produce changes i n the r a t i o s of the metabolic products formed (17). The purpose of d i s c u s s i n g the work on the metabolism of 22,25-dideoxyecdysone has been to show how organ c u l t u r e methodology has been a p p l i e d to the study of t h i s e c d y s t e r o i d that o f t e n behaves as a x e n o b i o t i c . Our r e s u l t s obtained by u s i n g organ c u l t u r e techniques so f a r agree q u i t e w e l l with

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what has been learned by using i n v i v o methods. However, the usefulness of t h i s methodology i s l i m i t e d somewhat by the expense i n both time and m a t e r i a l s compared with the use of l i v e i n s e c t s . In general, these i n v i t r o methods would appear to be more u s e f u l f o r the in-depth i n v e s t i g a t i o n of c r i t i c a l problem areas r a t h e r than f o r general usage. C e l l C u l t u r e Systems f o r Metabolic Studies The use of i n s e c t c e l l c u l t u r e systems f o r the study of the metabolism of x e n o b i o t i c s has not, to our knowledge, been attempted to date. The p e r i p h e r a l l i t e r a t u r e c o n s i s t s of only a few papers on the e f f e c t s of p e s t i c i d e s (32,33) and i n s e c t growth r e g u l a t o r s (34,35) on i n s e c t c e l l l i n e s and a s e r i e s of s t u d i e s on the e f f e c t s of v a r i o u s ecdysteroids on the morphology of c u l t u r e would l i k e to d i s c u s s b r i e f l u s i n g i n s e c t c e l l l i n e s f o r the study of the metabolism of xenobiotics. C e l l c u l t u r e s d i f f e r from organ c u l t u r e s p r i m a r i l y i n that they c o n s i s t of populations of d i v i d i n g i n d i v i d u a l c e l l s r a t h e r than h i g h l y organized t i s s u e s from a s i n g l e organism. As a r e s u l t , the l i f e of an organ c u l t u r e i s f i n i t e , while the l i f e of c e l l c u l t u r e s i s p o t e n t i a l l y i n f i n i t e (12). Primary c u l t u r e s that are derived d i r e c t l y from the donor may or may not develop i n t o s e l f r e p l i c a t i n g e s t a b l i s h e d c e l l l i n e s . Monolayer c u l t u r e s of f a t body c e l l s from pupae of l a r g e i n s e c t s such as the tobacco hornworm and embryonic c e l l s from the oothecae of grasshoppers and cockroaches have been s u c c e s s f u l l y prepared i n s u f f i c i e n t q u a n t i t i e s f o r experimental work (19) ( F i g . 6). However, because such small amounts of t i s s u e are a v a i l a b l e from most i n s e c t s and the contamination problems a r i s i n g from the use of l a r g e numbers of t i s s u e donors are so severe, the use of primary c u l t u r e s of i n s e c t c e l l s f o r experimental purposes o f f e r s few advantages over organ c u l t u r e s . The number of e s t a b l i s h e d l i n e s of i n s e c t c e l l s i s r a p i d l y i n c r e a s i n g , and many of these are a v a i l a b l e f o r experimental purposes (39). Advantages of C e l l Culture Systems The use of c e l l l i n e s f o r metabolic s t u d i e s has a number of inherent advantages and disadvantages (40). Perhaps the most obvious advantage i s that l a r g e amounts of uniform m a t e r i a l are a v a i l a b l e f o r experimentation. Large-scale r e p l i c a t i o n i s p o s s i b l e and there i s good sample u n i f o r m i t y when spinner f l a s k s (41) and r o l l e r b o t t l e s are used (42). The problems of s u r g i c a l shock and m i c r o b i o l o g i c a l contamination can be v i r t u a l l y e l i m i n a t e d ; and automatic devices f o r handling and counting c e l l s are a v a i l a b l e to keep labor at a minimum. C e l l l i n e s are a v a i l a b l e from a number of major d i p t e r a n , l e p i d o p t e r a n , and homopteran pests, although to

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(turc

Figure 6. Schematic of technique for preparing primary cell cultures from predorsal closure cockroach embryos. In preparing primary cultures of fat body cells, trypsinization is required to disperse the cells.

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date, none are a v a i l a b l e from eoleopteran or hymenopteran s p e c i e s (39). Disadvantages of C e l l C u l t u r e Systems At the present time, only a l i m i t e d number of c e l l l i n e s f o r which the t i s s u e of o r i g i n can be i d e n t i f i e d i s a v a i l a b l e . Even f o r these l i n e s , the evidence f o r i d e n t i t y i s weak (3). S k i l l f u l and determined e f f o r t s w i l l be necessary to develop new l i n e s from known t i s s u e s such as f a t body or to i d e n t i f y the t i s s u e of o r i g i n f o r present l i n e s . Another problem i s that w h i l e organ c u l t u r e s can be r e l a t e d back to i n d i v i d u a l i n s e c t s , c e l l l i n e s r e f l e c t the dynamics of l a r g e populations of i n d i v i d u a l s kept under constant s e l e c t i o n p r e s s u r e . The c e l l s must t h e r e f o r e be f r e q u e n t l y cloned and kept as f r o z e n stocks to maintain s t a b i l i t biochemical c h a r a c t e r i s t i c yet, very l i t t l e i s known about the metabolic c a p a b i l i t i e s of e x i s t i n g i n s e c t c e l l l i n e s and much remains to be done before such c e l l l i n e s can be considered u s e f u l t o o l s f o r the study of metabolism. At the present time we are working w i t h a c e l l l i n e (MRRL-CH-2) d e r i v e d from embryos of the tobacco hornworm (44). These c e l l s respond with a change i n morphology to the presence of 20-hydroxyecdysone at p h y s i o l o g i c a l c o n c e n t r a t i o n s and are capable of h y d r o x y l a t i n g the C-20 p o s i t i o n of aecdysone (43) (Table V ) . Other biochemical parameters of t h i s c e l l l i n e are c u r r e n t l y under i n v e s t i g a t i o n . Table V.

α-Ecdysone and 20-hydroxyecdysone present i n c e l l c u l t u r e s during i n c u b a t i o n of tobacco hornworm embryonic c e l l l i n e with 30 \xg of a-ecdysone.

Incubation Days

a-Ecdysone

20-Hydroxyecdysone

yg

Vtg

0 1 3 5 7

26.2 25.9 15.5 20.0 18.0

0.0 0.0 0.1 0.1 0.2

C e l l C u l t u r e s as a Source of S u b c e l l u l a r Components We have r e c e n t l y been u s i n g another tobacco hornworm embryonic c e l l l i n e (MRRL-CH-1) as a source of c e l l membrane f o r s t u d i e s of membrane t r a n s p o r t systems. As a r e s u l t of t h i s work i t has become apparent to us that c e l l l i n e s have

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some unique advantages as a source of subcellular components for various types of metabolism studies. We found that cell cultures can be subjected to bioassay and indeed to some biochemical procedures in the same medium in which they were grown (45). Thus, several of the hazards involved in tissue preparation such as osmotic shock and contamination with microorganisms are eliminated. Furthermore, large amounts of highly uniform material are available. Mammalian and plant tissue cultures are being used for such purposes (46,47), but few examples of such work with insect tissue cultures have been reported. (48). Concluding Remarks Insect organ cultures have proved to be extremely useful tools for studies of th dideoxyecdysone. The experimenta methodology and its use for the confirmation and extension of information obtained from in vivo studies make organ culture the method of choice for certain types of studies. The use of insect cell cultures for studying the metabolism of xenobiotics is s t i l l only in the early stages of development. Much work will have to be done before cell cultures can be considered useful tools for such studies. However, insect cell cultures are presently being used successfully to provide a highly uniform source of subcellular components for metabolic studies. Literature Cited 1. Marks, E. P., "Invertebrate Tissue Culture, Research Applications", (1976), Academic Press, NY, 177. 2. Agui, N., "Invertebrate Tissue Culture, Research Applications", (1976), Academic Press, NY, 133. 3. Landureau, C., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976), Academic Press, NY, 101. 4. Judy, K., Schooley, D., Dunhanm L., Hall, Μ., Bergot, J., and Siddall, J., Proc. Natl. Acad. Sci. U. S. Α., (1973) 70: 1509. 5. King, D., Bollenbacher, W., Borst, D., Vedeckis, W., O'Conner, J., Ittycheriah, P., and Gilbert, L . , Proc. Natl. Acad. Sci. U. S. Α., (1974) 71: 973. 6. Marks, E. P., Biol. Bull., (1971) 73: 83. 7. Agui, Ν., Appl. Entomol. Zool., (1974) 9: 256. 8. Wyatt, G. R., Chen, T., and Couble, T., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976), Academic Press, NY, 195. 9. Koeppe, J., and Offengand, J., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976), Academic Press, NY, 185.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

7. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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Marks, Ε. P., Biol. Bull., (1973) 145: 171. Marks, Ε. P., Biol. Bull., (1968) 135: 520. Federoff, S., In Vitro, (1966) 2: 155. Marks, E. P., and Holman, G., J. Insect. Physiol., (1974) 20: 2087. Schooley, D., Judy, Κ., Bergot, J . , Hall, Μ., and Siddall, J . , Proc. Natl. Acad. Sci. U. S. Α., (1973) 70: 2921. King. D. S., and Marks, E. P., Life Sci., (1974) 15: 147. Marks, E. P., Robbins, W. Ε., and Thompson, M. J., Lipids, (1978) 13: 259. Thompson, M. J., Marks, E. P., Robbins, W. E., Dutky, S. R., Finegold H. and F i l i p i P. Lipids in press Müller, P., Masner Life Sci. (1974) Marks, E. P., "Tissue Culture, Methods and Applications", (1973), Academic Press, NY, 153. Judy, K. and Marks, E. P., Gen. Comp. Endocrinol., (1971) 17: 351. Kaplanis, J. Ν., Robbins, W. Ε., Thompson, M. J., and Baumhover, Α., Η., Science, (1969) 166: 1540. Kaplanis, J. Ν., Dutky, S. R., Robbins, W. Ε., and Thompson, M. J., "Invertebrate Endocrinology and Hormonal Heterophylly", (1974), Springer-Verlag, NY, 161. Kaplanis, J. Ν., Thompson, M. J., Dutky, S. R., Robbins, W. Ε., and Lindquist, E., Steroids, (1972) 20: 105. Robbins, W. Ε., Kaplanis, J. Ν., Thompson, M. J., Shortino, T. J., and Joyner, S. C., Steroids, (1970) 16: 105. Robbins, W. Ε., Kaplanis, J. Ν., Thompson, M. J., Shortino, T. J., Cohen, C. F., and Joyner, S. C., Science, (1968) 161: 1158. Earle, N., Padovani, I., Thompson, M. J., and Robbins, W. E., J. Econ. Entomol., (1970) 63: 1064. Rose, G., Pomerat, C., Shindler, T., and Trunnel, J., J. Biophys. Biochem. Cytol., (1958) 4: 761. Marks, E. P., "Invertebrate Tissue Culture, Applications in Medicine, Biology and Agriculture", (1976) Academic Press, NY, 223. Svoboda, J . Α., Thompson, M. J., and Robbins, W. Ε., Lipids, (1972) 7: 553. Robbins, W. Ε., Thompson, M. J., Svoboda, J. Α., Shortino, T. J., Cohen, C. F., Dutky, S. R., and Duncan, O., Lipids, (1975) 10: 35. Marks, E. P., Gen. Comp. Endocrinol., (1973) 21: 472. Grace, T. D. C. and Mitsuhashi, J., "Arthropod Cell Cultures and Their Application to the Study of Viruses", (1971), Springer-Verlag, NY, 108.

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33. Mitsuhashi, J., Grace, T. D. C., and Waterhouse, D., Entomol. Exp. Appl., (1970) 13: 327. 34. Cohen, E. and Gilbert, L., J. Insect Physiol. (1973) 19: 1857. 35. Wyss, C., Experientia, (1976) 32: 1272. 36. Courgeon, Α. Μ., Exp. Cell Res., (1972) 75: 327. 37. Best-Belpomme and Courgeon, A. M., C. R. Acad. Sci., (1976) 283: 155. 38. Cherbas, P., Cherbas, L . , and Williams, C. Μ., Science, (1977) 197: 275. 39. Hink, W. F., "Invertebrate Tissue Culture, Research Applications", (1976), Academic Press, NY, 319. 40. Fry, J. R. and Bridges, J. W., "Progress in Drug Metabolism", (1977) John Wiley & Sons NY 71 41. Thayer, P. S., "Tissu (1973), Academi , , 42. Whittle, W. and Kruse, P., "Tissue Culture, Methods and Applications", (1973), Academic Press, NY, 327. 43. Marks, E. P. and Holman, G. Μ., In Vitro, (1978) in press. 44. Eide, P., Caldwell, J., and Marks, E. P., In Vitro, (1975) 11: 395. 45. Marks, E. P., In Vitro, (1978) 14: 374. 46. Gregor, H. D., Protoplasma, (1977) 91: 201. 47. North, H. and Menzer, R., Pestic. Biochem. Physiol., (1972) 2: 278. 48. Davis, J. and Hartig, W., Insect Biochem., (1977) 7: 77. RECEIVED

December 20,

1978.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8 The Use of Insect Subcellular Components for Studying the Metabolism of Xenobiotics C. F. W I L K I N S O N Department of Entomology, Cornell University, Ithaca, NY 14853

Our a b i l i t y to use to maximum advantage and i n r e l a t i v e s a f e t y the vast number of drugs, p e s t i c i d e s and other l i p o p h i l i c xenobiotics currently a extent on our a b i l i t y t organisms. Metabolic s t u d i e s i n mammals are e s s e n t i a l i n assessing the e f f i c a c y and s a f e t y of new drugs and data on the nature and t o x i c o l o g i c a l p r o p e r t i e s of p e s t i c i d e metabolites are mandatory i n e v a l u a t i n g the p o t e n t i a l hazard to man of residues of these m a t e r i a l s i n food or i n the environment. Comparative s t u d i e s with f i s h , b i r d s and other species are important i n determining the hazards posed to these animals by a large v a r i e t y of environmental p o l l u t a n t s . In a d d i t i o n to t h e i r d i r e c t importance i n safety/hazard e v a l u a t i o n metabolic s t u d i e s are b a s i c to our understanding of the mode of a c t i o n of b i o l o g i c a l l y a c t i v e m a t e r i a l s . They o f t e n y i e l d important information on enzymatic a c t i v a t i o n and d e t o x i c a t i o n processes and f r e q u e n t l y provide a mechanistic explanation of cases of s e l e c t i v e t o x i c i t y . I t i s i n the quest to o b t a i n a b e t t e r understanding of these processes and to u t i l i z e t h i s information i n the design of more e f f e c t i v e and s a f e r i n s e c t i c i d e s that has stimulated i n t e r e s t i n metabolic s t u d i e s i n i n s e c t s . As i s obvious from the p r e s e n t a t i o n s i n t h i s symposium, metabolic s t u d i e s may be c a r r i e d out i n v i v o i n the l i v i n g organism or i n v i t r o i n a v a r i e t y of preparations c o n s i s t i n g of i s o l a t e d organs, t i s s u e s , c e l l s or s u b c e l l u l a r components. In v i v o i n v e s t i g a t i o n s provide q u a n t i t a t i v e information on the overa l l r a t e of metabolism and on the nature of the t e r m i n a l metabol i t e s ; they seldom provide data on the nature of the metabolic intermediates or on the enzymatic mechanisms by which they are formed. In v i t r o experiments on the other hand permit the i d e n t i f i c a t i o n and study of i n d i v i d u a l r e a c t i o n mechanisms and products but are u s u a l l y of only l i m i t e d use i n e x p l a i n i n g the r a t e and p a t t e r n of metabolism i n the i n t a c t organism. C l e a r l y any complete metabolic study has to i n c l u d e both i n v i v o and i n v i t r o components; the s i t u a t i o n i s analogous to a puzzle where the 0-8412-0486-l/79/47-097-249$09.00/0 © 1979 A m e r i c a n C h e m i c a l Society

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i n v i v o s t u d i e s provide the pieces to the puzzle and the i n v i t r o s t u d i e s y i e l d the clues which enable the pieces to be put together i n a meaningful way. T h i s chapter w i l l address i t s e l f to the use of i n s e c t subc e l l u l a r components i n i n v i t r o s t u d i e s of the metabolism of x e n o b i o t i c s . Since most of the advantages and disadvantages of i n v i t r o s t u d i e s are of a general nature and l a r g e l y independent of the species employed, emphasis w i l l be given to d i s c u s s i n g some of the major problems encountered i n developing s u i t a b l e i n v i t r o systems and techniques with i n s e c t s p e c i e s . I t i s not the o b j e c t i v e of t h i s chapter to conduct a comprehensive survey of metabolic r e a c t i o n s with i n d i v i d u a l compounds or to d i s c u s s i n d e t a i l the c h a r a c t e r i s t i c s of the various enzymes i n v o l v e d i n x e n o b i o t i c metabolism. Those i n t e r e s t e d i n these areas are r e f e r r e d to some of th a v a i l a b l e (1-10), and th H i s t o r i c a l development The c a p a c i t y of i n s e c t s to metabolize s y n t h e t i c organic chemicals i n v i v o was f i r s t recognized during the l a t e 1940s when i t was discovered that metabolic d e h y d r o c h l o r i n a t i o n of DDT to the r e l a t i v e l y nontoxic DDE was a major causative f a c t o r i n the development of i n s e c t r e s i s t a n c e to t h i s i n s e c t i c i d e . In the years immediately f o l l o w i n g t h i s discovery i n v i v o i n v e s t i g a t i o n s with other i n s e c t i c i d e s i n c l u d i n g the c y c l o d i e n e s , the organophosphorus compounds and the carbamates revealed that almost a l l groups of compounds were s u s c e p t i b l e to metabolic attack by both i n s e c t s and a v a r i e t y of non-target organisms and that t h i s was o f t e n the dominant f a c t o r i n determining the degree and duration of t h e i r t o x i c a c t i o n . Recognition of the c r i t i c a l r o l e of metabolism i n r e l a t i o n to s e l e c t i v e t o x i c i t y and i n s e c t r e s i s t a n c e to i n s e c t i c i d e s gave f u r t h e r impetus to metabolic s t u d i e s and the intense a c t i v i t y of agro chemical i n d u s t r y during the 1950s provided a steady flow of new compounds with which to work. As a r e s u l t , i n v i v o metabolic s t u d i e s with i n s e c t s , mammals and other organisms assumed an ever important p o s i t i o n i n i n s e c t i c i d e research and were aided considerably by improved technology, p a r t i c u l a r l y the use of r a d i o a c t i v e t r a c e r s and the i n t r o d u c t i o n of more s o p h i s t i c a t e d instrumentation f o r the r e s o l u t i o n , d e t e c t i o n and i d e n t i f i c a t i o n of small amounts of metabolites. Since almost a l l of the e a r l y s t u d i e s with i n s e c t s were conducted i n v i v o only the end products of metabolism were observed. In most cases these were water s o l u b l e secondary conjugates which were d i f f i c u l t to i d e n t i f y and which provided l i t t l e or no information on the s t r u c t u r e s of the primary or intermediate metabolites from which they were derived or on the nature of the enzyme systems e f f e c t i n g the i n i t i a l a t t a c k on the parent compound. Indeed i n most of the e a r l y work these conjugates were simply c l a s s i f i e d as "water s o l u b l e s " and no f u r t h e r attempt was

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made to i d e n t i f y them. In s p i t e of these problems the importance of s e v e r a l primary metabolic r e a c t i o n s was w e l l e s t a b l i s h e d and i t was known that these could c a t a l y z e both the a c t i v a t i o n and d e t o x i c a t i o n of the parent compound. Dehydrochlorination was known to c o n s t i t u t e a major metabolic pathway f o r DDT and i t s d e r i v a t i v e s and h y d r o l y s i s and d e s u l f u r a t i o n were w e l l recognized r e a c t i o n s with a v a r i e t y of organophosphorus compounds. In a d d i t i o n the probable involvement of enzymatic o x i d a t i o n was s t r o n g l y i n d i c a t e d by the i d e n t i f i c a t i o n of metabolites such as epoxides, s u l f o x i d e s and sulfones from s e v e r a l compounds although the f u l l extent of t h i s involvement was not obvious. This then was the general s i t u a t i o n which p e r t a i n e d i n i n s e c t metabolic s t u d i e s up to and during the e a r l y 1950s. Somewhere around t h i s time there occurred a r a t h e r sudden r e a l i z a t i o n that the natur and mammals were e s s e n t i a l l r e f l e c t e d a b a s i c s i m i l a r i t y i n the enzymatic mechanisms i n v o l v e d . This tended to open up new and improved l i n e s of communication between i n d i v i d u a l s conducting metabolic s t u d i e s i n i n s e c t s and those working with mammals. The use of a v a r i e t y of i n v i t r o systems such as l i v e r s l i c e s , homogenates and s u b c e l l u l a r f r a c t i o n s had already proved u s e f u l i n mammalian s t u d i e s and i t was not long before the f i r s t attempts were made to develop s i m i l a r systems from i n s e c t s . E a r l y i n v i t r o s t u d i e s i n i n s e c t s u s u a l l y employed i n t a c t t i s s u e s or crude minces and homogenates. Although s e v e r a l organophosphorus e s t e r s were found to be hydrolyzed by the "aromatic e s t e r a s e " of the bee (Apis m e l l i f e r a ) abdomen (11) and malaoxon and acethion were shown to be degraded slowly by carboxylesterase a c t i o n i n cockroach ( P e r i p l a n e t a americana) minces and whole guts (12), homogenates of other i n s e c t s proved g e n e r a l l y i n a c t i v e towards a v a r i e t y of organophosphorus i n s e c t i c i d e s (13). Somewhat e a r l i e r than t h i s , i n t a c t i n s e c t t i s s u e s e s p e c i a l l y the gut had been found capable of a c t i v a t i n g schradan (14) and c a t a l y z i n g the o x i d a t i v e a c t i v a t i o n ( d e s u l f u r a t i o n ) of phosphorothionates such as parathion (15). Fenwick (16,17) was the f i r s t to demonstrate phosphoramidate a c t i v a t i o n i n a s u b c e l l u l a r preparation from l o c u s t ( S c h i s t o c e r c a gregaria) f a t body. He reported that 84% of the schradan o x i d i z i n g a c t i v i t y of a f a t body homogenate was i n the upper l a y e r of a heterogeneous 14,000g c e n t r i f u g a l sediment ( r e f e r r e d to somewhat questionably as the microsomal f r a c t i o n ) and that a c t i v i t y required the a d d i t i o n of e i t h e r the supernatant or exogenous NADPH. I t i s i n t e r e s t i n g that as a r e s u l t of these and r e l a t e d s t u d i e s Fenwick (17) as l a t e as 1958 found i t worthwhile to note that the l o c u s t f a t body homogenate contained " . . . (at l e a s t ) two d i s t i n c t types of p a r t i c l e s which resemble mammalian l i v e r mitochondria and microsomes." By the l a t e 1950s i t was w e l l e s t a b l i s h e d that i n mammals the primary metabolic attack on a l a r g e number of l i p o p h i l i c drugs and x e n o b i o t i c s was e f f e c t e d by a s e r i e s of o x i d a t i v e

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r e a c t i o n s a s s o c i a t e d with h e p a t i c microsomes (18). The f i r s t i n v i t r o demonstration of microsomal enzyme a c t i v i t y i n i n s e c t s was that of Agosin et a l . (19) who showed that NADPH-fortified microsomes prepared from whole german cockroaches ( B l a t t e l l a germanica) and other species c a t a l y z e d the h y d r o x y l a t i o n of DDT. This represented a milestone of s o r t s s i n c e i t provided the f i r s t r e a l i n d i c a t i o n that i n s e c t s contained an a c t i v e microsomal oxidase system s i m i l a r to that i n mammalian l i v e r and a l s o that i n v i t r o metabolic systems could be developed to reproduce the r e a c t i o n s o c c u r r i n g i n the l i v i n g i n s e c t . Since t h i s time i n v i t r o s t u d i e s employing s u b c e l l u l a r f r a c t i o n s derived from whole i n s e c t s or i n s e c t t i s s u e s have become almost r o u t i n e p r a c t i c e i n many l a b o r a t o r i e s . As a r e s u l t a great d e a l of information has been obtained on the types of metabolic r e a c t i o n s whic the general biochemical these r e a c t i o n s (1,6-10). I t has become c l e a r that as i n mammals, x e n o b i o t i c metabolism i n i n s e c t s i s accomplished by r e l a t i v e l y few general types of r e a c t i o n s . The importance of e s t e r cleavage by a s e r i e s of hydrolases (phosphatases, c a r b o x y l e s t e r a s e s , amidases, etc.) has been demonstrated with v a r i o u s substrates (20) and g l u t a t h i o n e t r a n s f e r a s e s are known to play important r o l e s i n primary r e a c t i o n s such as d e a l k y l a t i o n and d e a r y l a t i o n (21,22), d e c h l o r i n a t i o n (22,23,24) and thiocyanate metabolism (22,25). Epoxide hydratase a c t i v i t y i s known to be widely d i s t r i b u t e d i n i n s e c t s (20,26) and conjugating enzymes are known to c a t a l y z e numerous secondary r e a c t i o n s such as g l u c o s i d a t i o n and s u l f a t i o n (22). But perhaps most important of a l l i s c l e a r r e c o g n i t i o n of the dominance of the cytochrome P-450 mediated system i n i n s e c t s and i t s a b i l i t y to c a t a l y z e the primary metabolic a t t a c k on a l a r g e number of l i p o p h i l i c x e n o b i o t i c s through r e a c t i o n s such as epoxidation, h y d r o x y l a t i o n , N- and O-dealkyiation t h i o e t h e r o x i d a t i o n and d e s u l f u r a t i o n (1,8,9,10,27). What i s now emerging from comparative s t u d i e s i s a p i c t u r e of remarkable f u n c t i o n a l u n i t y with respect to the r e a c t i o n s of x e n o b i o t i c metabolism. There are of course some d i f f e r e n c e s between species as w i l l be discussed l a t e r i n t h i s symposium by Dr. T e r r i e r e but i n s p i t e of the dramatic v a r i a t i o n s i n s i z e , morphology, n u t r i t i o n , e c o l o g i c a l h a b i t a t and general l i f e s t y l e between say mammals and i n s e c t s , one cannot help but be impressed by the b a s i c s i m i l a r i t i e s which e x i s t at the s u b c e l l u l a r l e v e l . At the present time, however, the p i c t u r e i s s t i l l out of focus and much remains to be done, p a r t i c u l a r l y at the i n v i t r o l e v e l , before a more complete understanding can be achieved. General c o n s i d e r a t i o n s

r e l a t i n g to i n v i t r o

studies

The f i r s t major d e c i s i o n s which have to be made i n i n i t i a t i n g an i n v i t r o study are the species and l i f e stage of the i n s e c t to be employed. This w i l l be d i c t a t e d l a r g e l y by the purpose of the

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proposed study. In v i t r o metabolic s t u d i e s are u s u a l l y d i r e c t e d towards p r o v i d i n g i n f o r m a t i o n e i t h e r on the products of a p a r t i c u l a r r e a c t i o n ( s ) or on the biochemical character and mechanism of the enzyme which c a t a l y z e s the r e a c t i o n . In the former case, the i n v i t r o system i s used as a t o o l to produce primary or i n t e r mediary metabolites of a p a r t i c u l a r x e n o b i o t i c which may be r e q u i r e d to confirm the i d e n t i t y of t r a c e metabolites or metabolic pathways suggested from corresponding i n v i v o s t u d i e s ; i n the l a t t e r case s e l e c t e d x e n o b i o t i c s are used as t o o l s to c h a r a c t e r i z e the system which can then be employed as a source of more q u a n t i t a t i v e metabolic data or i n comparative s t u d i e s . If the proposed i n v e s t i g a t i o n i s concerned with e s t a b l i s h i n g the primary metabolites of some i n s e c t i c i d e i n a s p e c i f i c i n s e c t pest then c l e a r l y the specie mined and the l i f e stage a d u l t ) are presumably those at which the i n s e c t i c i d e i s d i r e c t e d i n the f i e l d , i . e . those causing the most economic damage. The major requirement here i s to develop an i n v i t r o system that w i l l reproduce metabolites observed i n v i v o . In t h i s type of study a d e t a i l e d knowledge of the system i s not of primary importance and, indeed, i n many cases the use of i n t a c t t i s s u e s or crude homogenates may be more u s e f u l than i n d i v i d u a l s u b c e l l u l a r components. If on the other hand the o b j e c t i v e of the study i s to charact e r i z e the enzymatic system r e s p o n s i b l e f o r a c e r t a i n type of metabolic r e a c t i o n , i t i s u s u a l l y d e s i r a b l e to work with more homogeneous, p u r i f i e d f r a c t i o n s and to give greater emphasis to o b t a i n i n g q u a n t i t a t i v e i n f o r m a t i o n . For s t u d i e s of t h i s type there i s c o n s i d e r a b l y more leeway i n the s e l e c t i o n of the s p e c i e s to be employed and i n view of the estimated existence of 2-10 m i l l i o n species of i n s e c t s , the t h e o r e t i c a l p o s s i b i l i t i e s are enormous. In p r a c t i c e , however, the choice of s p e c i e s i s u s u a l l y determined by a s e r i e s of convenience f a c t o r s which r e l a t e mainly to the amount of biomass a v a i l a b l e f o r study. T h i s i s probably the major l i m i t i n g f a c t o r i n most s t u d i e s on i n s e c t biochemistry. Consequently, wherever p o s s i b l e a r e l a t i v e l y l a r g e i n s e c t species should be s e l e c t e d . In many cases the l i f e c y c l e s of l a r g e i n s e c t s are q u i t e long so that i t i s o f t e n necessary to reach a compromise between s i z e and turnover time ( i . e . length of generat i o n ) . The species s e l e c t e d f o r study should be r e a d i l y amenable to mass r e a r i n g under l a b o r a t o r y c o n d i t i o n s (the a v a i l a b i l i t y of a s a t i s f a c t o r y a r t i f i c i a l d i e t i s advantageous) and should be a v a i l a b l e on a year-round b a s i s . C l e a r l y a species with only one generation per year or one with an o b l i g a t i v e p e r i o d of diapause would not be convenient f o r continuous study. I t i s as a r e s u l t of requirements of t h i s type that to date i n v i t r o s t u d i e s have been l i m i t e d to perhaps 30-40 i n s e c t species very few of which can be considered s e r i o u s pests of a g r i c u l t u r e or p u b l i c h e a l t h .

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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sources

Having s e l e c t e d an appropriate i n s e c t species and l i f e stage with which to work the next step i s t o decide on the t i s s u e source to be employed as a s t a r t i n g point f o r s u b c e l l u l a r f r a c t i o n a t i o n . Because of obvious l i m i t a t i o n s on the amount of t i s s u e u s u a l l y a v a i l a b l e , most of the e a r l y metabolic s t u d i e s were conducted with homogenates or s u b c e l l u l a r f r a c t i o n s derived from whole i n s e c t s . The use of whole-insect preparations i s s t i l l common i n s e v e r a l l a b o r a t o r i e s and can f r e q u e n t l y provide u s e f u l q u a l i t a t i v e information on x e n o b i o t i c metabolism. More o f t e n than not, however, enzyme a c t i v i t y i n such p r e p a r a t i o n s , p a r t i c u l a r l y that a s s o c i a t e d with the microsomal f r a c t i o n , i s very low or non­ e x i s t e n t and u s u a l l y bears l i t t l e resemblance to the true enzymatic c a p a b i l i t y o i n view of the heterogeneou i t i s r e a l l y q u i t e s u r p r i z i n g that such whole-insect preparations e x h i b i t any enzyme a c t i v i t y at a l l . C l e a r l y , the homogenization of whole i n s e c t s causes a t o t a l d i s r u p t i o n of t i s s u e and c e l l u l a r o r g a n i z a t i o n and r e s u l t s i n the r e l e a s e of a l a r g e number of endogenous m a t e r i a l s with p o t e n t i a l i n h i b i t o r y e f f e c t s on the enzyme under i n v e s t i g a t i o n . Several d i f f e r e n t types of endogenous i n h i b i t o r s have been encountered and i d e n t i f i e d i n the course of s t u d i e s with i n s e c t microsomes (8,10) and there i s no doubt that they o f t e n represent a s e r i o u s p r a c t i c a l problem i n i n v i t r o i n v e s t i g a t i o n s . The i n s e c t eye pigment, xanthommatin has been e s t a b l i s h e d as an important i n h i b i t o r y f a c t o r i n preparations from whole house f l i e s (28,29), f r u i t f l i e s (Drosophila melanogaster) and honey bees (Apis m e l l i f e r a ) (30). I t causes s u b s t a n t i a l i n h i b i t i o n of house f l y epoxidase a c t i v i t y a t concentrations as low as 5 χ 10" M and i n h i b i t o r y a c t i v i t y i s accompanied by a marked increase i n NADPH o x i d a t i o n (2_8,2_9). Studies on the mode of a c t i o n of xanthommatin have shown that i t accepts e l e c t r o n s from the f l a v i n , NADPH cytochrome c_ reductase, of the microsomal e l e c t r o n t r a n s p o r t chain thereby a c t i n g as an e l e c t r o n s i n k to impede the flow of reducing e q u i v a l e n t s to cytochrome P-450 (Figure 1) (28,29). The a b i l i t y of dihydroxanthommatin to undergo a u t o x i d a t i o n and to be o x i d i z e d i n the presence of cytochrome c_ or t y r o s i n a s e suggests the r a p i d regeneration of xanthommatin and a consequent enhance­ ment i n i t s i n h i b i t o r y p o t e n t i a l . Since xanthommmatin i s widely d i s t r i b u t e d as an i n s e c t eye pigment t h i s type of i n h i b i t i o n i s of p o t e n t i a l importance i n almost a l l preparations from whole insects. What may be a s i m i l a r type of i n h i b i t i o n has a l s o been encountered i n attempts to measure microsomal o x i d a t i o n i n prepa­ r a t i o n s from whole l a s t - i n s t a r lepidopterous l a r v a e j u s t p r i o r to pupation (31,32). In t h i s case, i n h i b i t i o n i s a s s o c i a t e d with s o l u b l e products (a v a r i e t y of quinones) of the m e l a n i z a t i o n or darkening process which i n v o l v e s the tyrosinase-mediated oxidation 7

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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XANTHOMMATIN

Components

DIH YDROXANTHOMMATIN

Auto oxidation Cytochrome c Tyrosinase

Drug Metabolism Reviews Figure 1.

Inhibition of microsomal oxidation by xanthommatin (S)

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XENOBIOTIC METABOLISM

of a v a r i e t y of ortho-dihydroxy compounds (e.g. DOPA) and t h e i r subsequent i n c o r p o r a t i o n i n t o the i n s e c t c u t i c l e . In l e p i d o p t e r ous l a r v a e , t h i s type of i n h i b i t i o n appears to be important only during l a t e l a r v a l development when the t y r o s i n a s e system i s a c t i v a t e d i n p r e p a r a t i o n f o r formation of the pupal coat. I t can be counteracted by the a d d i t i o n of l - p h e n y l - 2 - t h i o u r e a to the p r e p a r a t i o n s to i n h i b i t t y r o s i n a s e a c t i v i t y (31,32). A similar type of i n h i b i t i o n has a l s o been i m p l i c a t e d i n the i n s t a b i l i t y of mixed-function oxidase a c t i v i t y i n house f l y microsomes where the a d d i t i o n of cyanide (also a t y r o s i n a s e i n h i b i t o r ) has a marked s t a b i l i z i n g e f f e c t (33). In theory, quinones may be formed whenever c a t e c h o l s are brought i n t o contact with t y r o s i n a s e and s i n c e both are common i n i n s e c t s , workers should beware of preparations which become p r o g r e s s i v e l y darker on exposure to a i r Another major grou s e r i o u s l y impede i n v i t r those a s s o c i a t e d with the i n s e c t gut contents. Potent i n h i b i t o r s of microsomal o x i d a t i o n s have been reported i n the gut contents of several insect species including several lepidopterous larvae (34,35), a c a d d i s f l y l a r v a (Limnephilus sp.) (37), a sawfly l a r v a (Macremphytus varianus) (38) and the house c r i c k e t (Acheta domesticus) (38,39). The i n h i b i t o r y f a c t o r s i n the gut contents of the southern armyworm (Spodoptera e r i d a n i a ) (40) and the house c r i c k e t (39) have been p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d as p r o t e o l y t i c enzymes with molecular weights of 26,000 and 16,500 respectively. They are both undoubtedly n a t u r a l l y o c c u r r i n g d i g e s t i v e p r o t e i n a s e s q u i t e s i m i l a r to t r y p s i n and l i k e t r y p s i n t h e i r i n h i b i t o r y a c t i o n r e s u l t s from a d i r e c t p r o t e o l y t i c a t t a c k on the microsomal p r o t e i n (39,41). The e f f e c t of these proteases on the microsomes i s q u i t e s p e c i f i c i n that they cause the s o l u b i l i z a t i o n of the f l a v o p r o t e i n , NADPH cytochrome c_ reductase, and consequently d i s r u p t e l e c t r o n flow to cytochrome P-450 (39,41) (Figure 2 ) . There appears to be no e f f e c t on cytochrome b$ of P-450 and no e f f e c t on s u b s t r a t e b i n d i n g to the l a t t e r (41) so i t appears that the f l a v o p r o t e i n i s the major t a r g e t , p o s s i b l y due to i t s v u l n e r a b l e l o c a t i o n on the outer surface of the membrane. Although s i m i l a r i n t h e i r o v e r a l l e f f e c t on the microsomes, the armyworm and c r i c k e t gut content m a t e r i a l s e x h i b i t p r o p e r t i e s i n d i c a t i n g they are not i d e n t i c a l (Table 1). Thus i n c o n t r a s t to the armyworm m a t e r i a l which shows a s i m i l a r i n h i b i t o r y e f f e c t on i n s e c t and mammalian l i v e r microsomes the c r i c k e t m a t e r i a l i s much l e s s a c t i v e towards the l a t t e r and while both appear to be s e r i n e p r o t e i n a s e s s u s c e p t i b l e to i n h i b i t i o n by phenylmethanes u l f o n y l f l u o r i d e (PMSF) the c r i c k e t but not the armyworm m a t e r i a l i s s e n s i t i v e to soy t r y p s i n i n h i b i t o r . Bovine serum albumin (BSÀ) has a marked p r o t e c t i v e e f f e c t on microsomes i n the presence of the armyworm gut i n h i b i t o r whereas t h i s i s observed with the c r i c k e t m a t e r i a l only at high l e v e l s of BSA (39). The p r o t e c t i v e a c t i o n of BSA against at l e a s t some of these p r o t e o l y t i c enzymes undoubtedly accounts f o r the enhanced microsomal enzyme a c t i v i t y

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

WILKINSON

Figure 2.

Insect Subcellular

Components

Solubilization of NADPH-Cytochrome c reductase by gut contents inhibitor of southern armyworm (S. eridania) (41).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

258

XENOBIOTIC METABOLISM

TABLE I Properties

of gut content i n h i b i t o r s from a/ S. e r i d a n i a and A. domesticus— Source of i n h i b i t o r

Property

S. e r i d a n i a

A.

Molecular weight

26,000

16,500

Yes

Yes

Proteolytic activity

(casein)

Mg

2+ , , Ca (mM)

Soy

t r y p s i n i n h i b i t o r (lmg/inc.)

w

2+

domesticus

x

Stimulate

effec

No e f f e c t (F )

Inhibition (F )

Inhibition

Inhibition

Armyworm midgut

0.97

0.17

Mammalian l i v e r

^2.0 (F^)

2

PMSF

X

I n h i b i t i o n epoxidase (I^Q, mg/inc.)

(mouse)

7.0 (F ) X

(rat)

Reversal of microsomal i n h i b i t i o n BSA PMSF Soy

(0.5-1.0mM) t r y p s i n i n h i b i t o r (lmg/inc.)

Yes

No ( s l i g h t )

Yes

Yes Yes

— Data from K r i e g e r and Wilkinson (40) and B r a t t s t e n and W i l k i n s o n (39). F^ i s crude f r e e z e - d r i e d gut contents s o l u b l e f r a c t i o n ; F2 i s f r e e z e - d r i e d m a t e r i a l a f t e r passage through Sephadex G-50 or G-100.

observed f o l l o w i n g a d d i t i o n of t h i s m a t e r i a l to homogenates of whole house f l i e s (42). C a s e i n o l y t i c a c t i v i t y has been reported i n whole i n s e c t homogenates of s e v e r a l species (40) and i t i s l i k e l y that p r o t e o l y t i c enzymes of t h i s type are of broad general significance i n i n v i t r o studies i n insects. I t should be obvious from t h i s d i s c u s s i o n that a v a r i e t y of endogenous i n h i b i t o r s l i b e r a t e d during homogenization make whole i n s e c t s a g e n e r a l l y poor t i s s u e source f o r i n v i t r o s t u d i e s p a r t i c u l a r l y those concerned with the biochemical c h a r a c t e r i z a t i o n

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8.

WILKINSON

Insect Subcellular

Components

259

of the enzymes involved i n metabolism. In some eases, however, due to f a c t o r s such as very small s i z e (e.g. mosquitoes or aphids) or l i m i t e d a v a i l a b i l i t y of the species i n question the use of whole i n s e c t preparations i s unavoidable and can o f t e n be u s e f u l i n gross metabolic s t u d i e s . In t h i s case compromises must be made. The chances of encountering endogenous i n h i b i t o r s are, of course, considerably decreased when s p e c i f i c body regions of the i n s e c t are employed. Thus the use of house f l y abdomens has proved advantageous over whole house f l i e s (8,9,43) ( s i n c e i t immediately avoids the problems a s s o c i a t e d with xanthommatin) and ingenious methods have been developed f o r the mass separation of i n s e c t body segments. Further improvement may be achieved by the a d d i t i o n of BSA (39), cyanide (33) or other p r o t e c t i v e agents to the preparations although care must be take not having other e f f e c t In general, the c l e a r message i s that wherever the s i z e of the i n s e c t permits, i n d i v i d u a l t i s s u e s or organs should be employed as a t i s s u e source f o r homogenization and s u b c e l l u l a r f r a c t i o n a t i o n . Unfortunately, t i s s u e d i s s e c t i o n i s o f t e n a tedious process and there i s u s u a l l y no way to avoid the i n d i v i dual handling of l a r g e numbers of i n s e c t s . The most convenient procedures f o r t h i s vary considerably from one species to another. As a r e s u l t of considerable work i n recent years, i t has been e s t a b l i s h e d that the patterns of t i s s u e d i s t r i b u t i o n of microsomal oxidase a c t i v i t y vary considerably both between d i f f e r e n t orders of i n s e c t s and between species i n a s i n g l e order. In general, however, the t i s s u e s found to be most a c t i v e are various p o r t i o n s of the alimentary t r a c t , the f a t body or the Malpighian tubules (Figure 3) (8). In lepidopterous l a r v a e maximum oxidase a c t i v i t y i s a s s o c i a t e d with the gut t i s s u e s , p a r t i c u l a r l y those of the midgut (Figure 4) (34). This d i s t r i b u t i o n p a t t e r n has been demonstrated with approximately 40 d i f f e r e n t species (44,45) the only exception to date being the cabbage looper ( T r i c h o p l u s i a n i ) where preparat i o n s from the f a t body are more a c t i v e than those from the gut (35). High t i t e r s of microsomal oxidase a c t i v i t y have a l s o been reported i n the gut t i s s u e s of various other i n s e c t s (J3) although i n many cases t h i s i s a s s o c i a t e d with a much broader p a t t e r n of t i s s u e d i s t r i b u t i o n as i n various orthopteran s p e c i e s . In s e v e r a l species of cockroach epoxidation and h y d r o x y l a t i o n a c t i v i t y occur i n v a r i o u s t i s s u e s i n c l u d i n g the gut, f a t body and Malpighian tubules the r e l a t i v e a c t i v i t y of each v a r y i n g with the species (Figure 4B,C) (8). In the house c r i c k e t (Acheta domesticus) maximum a c t i v i t y i s found i n the Malpighian tubules (Figure 4D) (38). As i n mammals, t h e r e f o r e , microsomal oxidase a c t i v i t y i n i n s e c t s i s found i n s e v e r a l d i f f e r e n t t i s s u e s and the observed patterns may r e f l e c t a s t r a t e g i c l o c a l i z a t i o n l i n k e d with the major p o r t a l s of entry of x e n o b i o t i c s i n t o the organism (8,27).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

260

FOREGUT

"V

HEAD

"

Figure 3.

MIDGUT

ν

V

THORAX

ABDOMEN

Insect tissues involved in microsomal oxidation

FG

GC

M G H G

MT

F Β

Figure 4. Distribution of microsomal epoxidase activity in insect tissues: (A) southern armyworm (S. eridania) (34); (B) Madagascar cockroach (G. portentosa) (C) American cockroach (P. americanum) (48); (D) house cricket (A. domesticus) (38). Solid bars represent specific activity (per mg protein); striped bars represent activity per insect.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8.

WILKINSON

Insect Subcellular

261

Components

Thus the gut t i s s u e s may c o n s t i t u t e the major s i t e f o r metabolism of x e n o b i o t i c s ingested i n the food whereas the f a t body and/or Malpighian tubules p o s s i b l y p l a y a greater r o l e i n the metabolism of m a t e r i a l s e n t e r i n g the i n s e c t by d i r e c t integumental penetrat i o n . This can be viewed as analogous to the d i s t r i b u t i o n of microsomal o x i d a t i o n i n the s k i n , lung, gut and l i v e r of mammals where they appear to be s t r a t e g i c a l l y l o c a t e d to act as the f i r s t l i n e of defense against x e n o b i o t i c s . In a s s e s s i n g the r e l a t i v e metabolic importance of a given t i s s u e to the i n t a c t i n s e c t , i t i s important to consider not only the s p e c i f i c a c t i v i t y of the enzyme i n a homogenate or s u b c e l l u l a r f r a c t i o n ( i . e . a c t i v i t y / m g p r o t e i n ) but a l s o the r e l a t i v e biomass of the v a r i o u s t i s s u e s . Consequently, a t i s s u e of l a r g e biomass but low s p e c i f i c oxidase a c t i v i t y may be more important i n i t s o v e r a l l metabolic c a p a c i t specific activity. Thi patterns of d i s t r i b u t i o n of oxidase a c t i v i t y based on measurements of s p e c i f i c a c t i v i t y with those c a l c u l a t e d on a per i n s e c t b a s i s (Figure 4). Even when working with i n d i v i d u a l t i s s u e s , i t i s important to be aware of the p o s s i b i l i t y of encountering endogenous enzyme i n h i b i t o r s . These can o f t e n be detected by combining homogenates of d i f f e r e n t t i s s u e s and examining the a c t i v i t y data f o r i n h i b i t o r y e f f e c t s . As shown i n Table 2, the t o t a l a c t i v i t i e s of combinations of homogenates of v a r i o u s armyworm t i s s u e s are equal to the sum of t h e i r i n d i v i d u a l a c t i v i t i e s and, t h e r e f o r e , can be assumed to be f r e e of i n h i b i t o r y f a c t o r s . In other cases, the

TABLE 2 Epoxidase a c t i v i t i e s of armyworm midgut homogenate alone and i n presence of homogenates of other t i s s u e s

Protein (mg/incubât ion)

Tissue

Epoxidase a c t i v i t y (nmoles/10 min) Observed Calculated

2.1

14.8

Foregut Foregut + MG

1.0 3.1

0.5 15.1

15.3

Hindgut Hindgut + MG

1.2 3.3

0.6 15.6

15.4

Fat body Fat body + MG

6.6 8.7

3.3 17.8

18.1

Malpighian tubules Malpighian tubules + MG

1.0 3.1

2.2 16.8

17.0

Midgut

(MG)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

262

a c t i v i t i e s of homogenates or s u b c e l l u l a r f r a c t i o n s of t i s s u e s can be compared with that of the i n t a c t t i s s u e . In the case of adult worker honey bees, epoxidase a c t i v i t y of i n t a c t midguts was decreased approximately 90% simply by opening the gut by l o n g i t u d i n a l i n c i s i o n and was t o t a l l y l o s t f o l l o w i n g homogenization of the t i s s u e (Table 3) (46). Subsequently a potent i n t r a c e l l u l a r i n h i b i t o r y f a c t o r was i s o l a t e d and p a r t i a l l y p u r i f i e d from the s o l u b l e f r a c t i o n of the gut (47). I n h i b i t i o n was a s s o c i a t e d with the n u c l e i c a c i d (RNA) moiety of a macromolecule ( p o s s i b l y a n u c l e o p r o t e i n ) with a molecular weight of approximately 19,000 and could be reversed by d i g e s t i o n with r i b o n u c l e a s e (RNase T^ of TABLE 3 Epoxidase a c t i v i t honey bee (adul Midgut treatment

) midgu Epoxidase a c t i v i t y pmole/min/mg wet. wt.

Intact

1.31

Opened

0.14

Homogenized

Not d e t e c t a b l e

Data from G i l b e r t and Wilkinson (46)

p a n c r e a t i c RNase) (Table 4). F o l l o w i n g t h i s f i n d i n g , i t was d i s covered that s e v e r a l commercially a v a i l a b l e n u c l e i c a c i d s i n c l u d i n g core and t r a n s f e r RNA from baker's yeast and t r a n s f e r RNA from T o r u l a yeast a l s o i n h i b i t e d i n s e c t (armyworm gut) microsomal oxidases but had l i t t l e or no e f f e c t on those from mammalian l i v e r (47). Although to date t h i s i s the only i n h i b i t o r of t h i s type r e p o r t e d , i t i s p o s s i b l e that others w i l l be found i n v a r i o u s insect preparations. Another important parameter which has to be considered i n the s e l e c t i o n of a s u i t a b l e source of t i s s u e f o r i n v i t r o i n v e s t i g a t i o n i s the age or l i f e stage o f the i n s e c t to be used. I t i s now w e l l e s t a b l i s h e d that the a c t i v i t y of the enzymes i n v o l v e d i n x e n o b i o t i c metabolism change d r a m a t i c a l l y with age and stage of development i n v a r i o u s i n s e c t s p e c i e s ( 8 ) . T h i s i s c l e a r l y i l l u s t r a t e d by the p a t t e r n s of microsomal oxidase a c t i v i t y shown i n F i g u r e 5. In g e n e r a l , microsomal oxidase a c t i v i t y i s only found i n a c t i v e l y feeding l i f e stages of the i n s e c t . Thus maximum a c t i v i t y i s u s u a l l y a s s o c i a t e d with the l a r v a l or nymphal stages of development and i n the a d u l t s where these feed; i n s e c t eggs and pupae are u s u a l l y devoid of oxidase a c t i v i t y . In southern armyworm l a r v a e , microsomal o x i d a t i o n a c t i v i t y ( s p e c i f i c a c t i v i t y ) i n

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

WILKINSON

Insect Subcelluhr

Age

(weeks)

Components

263

littar

(tine i i weeks)

Drug Metabolism Reviews Figure 5. Age-activity profiles of microsomal epoxidation in (A) southern armyworm (S. eridania) (34); (B) house cricket (A. domesticus) (38); and (C and D) Madagascar cockroach (G. portentosa) (48).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

264 TABLE 4

E f f e c t of RNase T^ and p a n c r e a t i c RNase on of the honey bee midgut

inhibitor Reversal of i n h i b i t i o n (%)

Enzyme (unit) RNase Τ -1

activity

0 24 100

(5) (260) (2800)

P a n c r e a t i c RNase

(0.072)

14

(0.360)

50

Data from G i l b e r t and Wilkinson

(47)

the gut increases about 30-fold during development from the f o u r t h to s i x t h l a r v a l i n s t a r (Figure 5A) and shows an e q u a l l y dramatic d e c l i n e as the l a r v a e terminate feeding and prepare f o r pupation (34); a s i m i l a r p a t t e r n has been observed with s e v e r a l other species (44). The increase i n a c t i v i t y does not occur i n a continuous manner, however, but shows a marked decrease during each l a r v a l molt (dotted l i n e s Figure 5A). Studies with other i n s e c t species have emphasized the remarkable patterns of oxidase a c t i v i t y which occur during i n s e c t development p a r t i c u l a r l y those o c c u r r i n g during the l a r v a l or nymphal molts. Thus a l d r i n epoxidation a c t i v i t y i n the Malpighian tubules of the house c r i c k e t i s low during the molts from 8th to 9th nymphal i n s t a r and from the 9th i n s t a r to the adult and then increases as the adult matures (Figure 5B) (38). T h i s rhythmic developmental c y c l e becomes much c l e a r e r i n the midgut-caeca p r e p a r a t i o n from the Madagascar cockroach (Figure 5C and D) where i t i s q u i t e evident that oxidase a c t i v i t y i s low during the molt and passes through a maximum about midway through each i n s t a r (48). The r e s u l t s of these and other i n v e s t i g a t i o n s s t r o n g l y suggest that microsomal oxidase a c t i v i t y i s under s t r i c t metabolic c o n t r o l and that t h i s i s c l o s e l y l i n k e d with the process of metamorphosis. From a p r a c t i c a l viewpoint these r a p i d and dramatic changes i n enzyme a c t i v i t y can provide a r e a l headache i n i n v i t r o s t u d i e s . Researchers i n t h i s f i e l d would be w e l l advised to e i t h e r e s t a b l i s h the a c t i v i t y patterns i n the i n s e c t species with which they are working or to avoid working with i n s e c t s c l o s e to the molt. In some types of i n v e s t i g a t i o n s (e.g. enzyme i n d u c t i o n s t u d i e s ) where comparisons between t r e a t e d and c o n t r o l groups are r e q u i r e d , i t i s e s s e n t i a l to use groups of i n s e c t s c l o s e l y matched

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8.

WILKINSON

Insect Subcelluhr

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265

(+2h) with respect to age (49). Procedures f o r o b t a i n i n g i n s e c t s u b c e l l u l a r components As with other t i s s u e s , the p r e p a r a t i o n of s u b c e l l u l a r components from i n s e c t s i s e f f e c t e d by two successive steps: homogenization and f r a c t i o n a t i o n . In general, the methods which have been employed are based on procedures which have proved s u c c e s s f u l f o r mammalian t i s s u e s . However, due to l a r g e v a r i a t i o n s i n the nature and morphological heterogeneity of the i n s e c t t i s s u e s employed, numerous m o d i f i c a t i o n s have been made i n attempts to optimize the procedures. These have been discussed at some length i n previous reviews (8,9,10). Various procedures have been described f o r the homogenization of whole i n s e c t s , i n s e c and u s u a l l y the method o process of t r i a l and e r r o r f o r any given t i s s u e source. Homogeniz a t i o n i s u s u a l l y achieved with e i t h e r a mechanical blender, a hand-operated or motor-driven t i s s u e grinder or a mortar and p e s t l e (8,9,10). There i s a general concensus of opinion that the procedure should be as gentle as p o s s i b l e and yet s u f f i c i e n t l y d r a s t i c to s a t i s f a c t o r i l y d i s r u p t c e l l s i n heterogeneous t i s s u e s o f t e n c o n t a i n i n g s c l e r o t i z e d c u t i c l e , muscle f i b e r s , e t c . A l l operations should be c a r r i e d out at 0-2°C. Mechanical blenders (Waring blender, Omni-mixer, etc.) have been used f o r l a r g e batches of whole i n s e c t s or i n s e c t body regions (e.g. house f l i e s or house f l y abdomens) but i n general, oxidase a c t i v i t y i n microsomal f r a c t i o n s derived from such homogenates i s considerably lower than i n those from homogenates prepared with a P o t t e r Elvehjem t i s s u e grinder (43,50) or by a gentle pounding a c t i o n i n a mortar (10,51). T h i s l o s s of enzyme a c t i v i t y may r e s u l t i n part from denaturation through excessive abrasive a c t i o n of hard c h i t i n o u s p o r t i o n s of the i n s e c t or by the more e f f i c i e n t r e l e a s e of endogenous i n h i b i t o r s . V i o l e n t mechanic a l blending undoubtedly a i d s i n the r e l e a s e of the s p a r i n g l y s o l u b l e xanthominatin i n p r e p a r a t i o n s c o n t a i n i n g i n s e c t heads and there are reports that microsomes from blended whole house f l i e s contain a b-type cytochrome (probably cytochrome oxidase) which i s a s s o c i a t e d with t h o r a c i c sarcosomes or sarcosomal fragments and which i n t e r f e r e s with s p e c t r a l determination of microsomal cytochrome P-450 (10,51,52). The l a t t e r i s not observed i n house f l y homogenates prepared by the s o - c a l l e d "mortar" procedure (10,51). The degree or d u r a t i o n of homogenization of whole i n s e c t s or body regions i s a l s o important even when a t i s s u e grinder i s employed. U s u a l l y only a few passes of the plunger are required and b e t t e r r e s u l t s are achieved by use of a r e l a t i v e l y l o o s e - f i t t i n g p e s t l e of T e f l o n or smooth g l a s s . The homogenization of i n d i v i d u a l t i s s u e s u s u a l l y presents fewer problems and here a v a r i e t y of hand-operated or motord r i v e n t i s s u e g r i n d e r s provide the best r e s u l t s . The small

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amounts of t i s s u e a v a i l a b l e as w e l l as the heterogenous o f t e n " s t r i n g y " nature of the t i s s u e (e.g. armyworm gut) u s u a l l y make mechanical blenders q u i t e i n e f f e c t i v e f o r homogenization purposes. Homogenization with a t i s s u e grinder should be as gentle and b r i e f as p o s s i b l e and again T e f l o n or smooth glass p e s t l e s are p r e f e r able to those of ground g l a s s . Several d i f f e r e n t types of homogenization media have been employed by v a r i o u s workers and obviously can have an e f f e c t on the f i n a l enzymatic a c t i v i t y of the preparation. Although there are reports that the i o n i c strength of the medium may be important (43) the most commonly used media are 0.15 M KC1 or 0.25 M sucrose which may or may not be b u f f e r e d at various pH l e v e l s (8,9,10). O c c a s i o n a l l y BSA (1-2% w/v) or other m a t e r i a l s such as cyanide, phenylmethanesulfonyl f l u o r i d e , e t c . may be added to at l e a s t p a r t i a l l y counteract th (8,42,43). S u b c e l l u l a r f r a c t i o n a t i o n of homogenates of whole i n s e c t s or i n s e c t t i s s u e s i s u s u a l l y achieved by the technique of d i f f e r e n t i a l c e n t r i f u g a t i o n , f i r s t pioneered by A l b e r t Claude with mammal i a n l i v e r c e l l s and subsequently widely a p p l i e d to many other t i s s u e s (53). B r i e f l y the technique i n v o l v e s c e n t r i f u g i n g the i n i t i a l homogenate at about l,000xg f o r 10 minutes to remove n u c l e i and l a r g e r c e l l d e b r i s followed by a 9,000-12,000xg c e n t r i f u g a t i o n f o r 10-30 minutes to sediment mitochondria and other intermediate s i z e d p a r t i c l e s and c e l l o r g a n e l l e s . Further c e n t r i f u g a t i o n of the postmitochondrial supernatant f o r 60 minutes at 100,000-200,OOOxg y i e l d s the s o - c a l l e d microsomal f r a c t i o n and a " s o l u b l e " supernatant. With i n s e c t t i s s u e s t h i s general procedure i s often modified and the a c t u a l c e n t r i f u g a l c o n d i t i o n s ( f o r c e , time) u s u a l l y vary somewhat between d i f f e r e n t l a b o r a t o ries. The procedure may a l s o i n c l u d e f i l t r a t i o n of the o r i g i n a l b r e i through cheese c l o t h , cotton or g l a s s wool to remove the o f t e n s u b s t a n t i a l amounts of c h i t i n o u s debris and l i p i d s r e l e a s e d during homogenization. The a p p l i c a t i o n of t h i s technique to the s u b c e l l u l a r f r a c t i o n a t i o n of i n s e c t t i s s u e homogenates i s based on the l a r g e l y unproven assumption that the s u b c e l l u l a r o r g a n e l l e s and membrane p a r t i c l e s derived from i n s e c t t i s s u e s e x h i b i t s i m i l a r sedimentat i o n c h a r a c t e r i s t i c s to those from mammalian l i v e r c e l l s . In view of the h i g h l y heterogeneous nature of most of the i n s e c t t i s s u e courses used f o r s t u d i e s of x e n o b i o t i c metabolism t h i s i s a dangerous assumption which should be only made with extreme care. Moreover the procedures found s a t i s f a c t o r y f o r one t i s s u e may not be a p p l i c a b l e to another. S u b c e l l u l a r f r a c t i o n s can be c l a s s i f i e d morphologically by means of l i g h t or e l e c t r o n microscopy as containing n u c l e i , mitochondria, microsomal p a r t i c l e s (from the membranous endoplasmic reticulum) e t c . In a d d i t i o n , the discovery that c e r t a i n types of enzymes are u s u a l l y a s s o c i a t e d with a s i n g l e c l a s s of p a r t i c l e s has l e d to the development of a s e r i e s of "marker" enzymes which

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can be used to c l a s s i f y the various s u b c e l l u l a r f r a c t i o n s i n b i o ­ chemical terms. Few attempts have yet been made to c h a r a c t e r i z e i n s e c t s u b c e l l u l a r f r a c t i o n s e i t h e r morphologically or biochemi­ c a l l y and most workers i n t h i s f i e l d s t i l l seem content to a s s o c i a t e t h e i r enzyme a c t i v i t i e s with s u b c e l l u l a r f r a c t i o n s defined only by values obtained from the u l t r a c e n t r i f u g e . L i t t l e a t t e n t i o n i s a l s o given to c a l c u l a t i n g a complete balance sheet of the t o t a l enzyme a c t i v i t y and p r o t e i n concentration i n each f r a c ­ t i o n and comparing these with values f o r the o r i g i n a l homogenate. Thus i n the absence of t o t a l p r o t e i n concentration the high s p e c i f i c a c t i v i t y of an enzyme i n some s u b c e l l u l a r f r a c t i o n y i e l d s l i t t l e information with regard to i t s o v e r a l l d i s t r i b u t i o n and a meaningful d i s c u s s i o n of s u b c e l l u l a r f r a c t i o n a t i o n i s d i f f i c u l t . From the s t u d i e s which have been conducted i t i s reasonable to conclude that as i n metabolism i n i n s e c t s ar or the microsomal f r a c t i o n s of the c e l l . Although i t i s p o s s i b l e that problems could be encountered by the adsorbance of s o l u b l e enzymes onto l a r g e r c e l l o r g a n e l l e s , the preparation of s a t i s f a c ­ tory s o l u b l e enzyme f r a c t i o n s does not appear to have been a serious problem to date. Most a t t e n t i o n has been focussed on s u b c e l l u l a r f r a c t i o n a t i o n procedures to obtain s a t i s f a c t o r y microsomal f r a c t i o n s from i n s e c t t i s s u e s . In s e v e r a l cases, microsomal f r a c t i o n s e x h i b i t i n g high l e v e l s of drug and i n s e c t i c i d e o x i d i z i n g a c t i v i t y can be prepared by a d i f f e r e n t i a l c e n t r i f u g a t i o n procedure s i m i l a r to that used f o r mammalian l i v e r . Thus i n the case of the armyworm (Spodoptera e r i d a n i a ) l a r v a l gut preparation, the microsomal f r a c t i o n (100,000xg, 60 minutes) i s c l e a r l y the major i n t r a c e l l u l a r l o c a ­ t i o n of the enzymes c a t a l y z i n g epoxidation and N-demethylation (Figure 6A and Β r e s p e c t i v e l y ) and Figure 6B shows that the d i s t r i b u t i o n pattern f o r N-demethylase a c t i v i t y i s e s s e n t i a l l y i d e n t i c a l to that f o r NADPH-cytochrome c_ reductase a recognized microsomal marker enzyme (54) ; that t h i s f r a c t i o n i s derived mainly from the endoplasmic r e t i c u l u m has been obtained by e l e c t r o n microscopy (55). In other cases, however, the preparation of i n s e c t microsomes by d i f f e r e n t i a l c e n t r i f u g a t i o n has proved much more d i f f i c u l t . One of the most common problems which i s encountered i s that most of the x e n o b i o t i c o x i d i z i n g a c t i v i t y associated with the micro­ somal f r a c t i o n i s sedimented at unexpectedly low g-forces (8). T h i s has been reported f o r f a t body preparations from the American cockroach ( P e r i p l a n e t a americana) (56,57), and blowfly l a r v a ( C a l l i p h o r a erythrocephala) (58) and homogenates of c r i c k e t (Acheta domesticus) Malpighian tubules (38) and Madagascar cock­ roach (Gromphadorhina portentosa) gut-caeca t i s s u e s (48). Thus i n the l a t t e r example approximately 48% of the a l d r i n epoxidase a c t i v i t y was sedimented during a 15 minute s p i n at 12,000xg (Figure 6C) using the t y p i c a l d i f f e r e n t i a l c e n t r i f u g a t i o n proce­ dure. I f , instead the crude homogenate ( i n 0.25M sucrose

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Figure 6.

Subcelluhr fractionation of microsomal oxidase activity from insect tissues.

A and Β are epoxidation (34) and Ν-demethylation (54) activities, respectively, in south­ ern armyworm midgut tissues; dotted line in Β is NADPH-cytochrome c reductase activity. M is 10,000-12,000 g X 10-15 min pellet, Ρ is 105,000 g X 60-90 min pellet, S is supernatant. C and D are epoxidation activity from midgut caeca tissue of Mada­ gascar cockroach (48).

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containing 50mM T r i s - H C l , pH 7.4) i s layered over 1.6M sucrose and c e n t r i f u g e d f o r 45 minutes at 150,000xg i n a swinging bucket r o t o r two major p a r t i c u l a t e f r a c t i o n s are obtained a dense p e l l e t ( P ) which sediments to the bottom of the tube and a l i g h t p r o t e i n Band (P-, ) at the sucrose i n t e r f a c e . The l a t t e r which represents about 28% of the t o t a l p r o t e i n contains 90.4% of the t o t a l epoxidase a c t i v i t y (Figure 6D) has been shown by e l e c t r o n microscopy t o c o n s i s t l a r g e l y of membranous v e s i c l e s from the endoplasmic reticulum. A s i m i l a r procedure i n v o l v i n g sucrose density gradient c e n t r i f u g a t i o n has been found u s e f u l i n p r e p a r i n g microsomal f r a c t i o n s from c r i c k e t Malpighian tubules (38) and honey bee l a r v a l gut t i s s u e s (46). It t h e r e f o r e appears that under c e r t a i n c o n d i t i o n s , the microsomal p a r t i c l e s i n homogenates of some i n s e c t t i s s u e s e i t h e r aggregate or become adsorbe such as mitochondria. Worker t h i s p o s s i b i l i t y and where necessary modify t h e i r procedures a c c o r d i n g l y . The suspension medium may have some e f f e c t on t h i s behavior s i n c e i t can modify the physicochemical forces i n v o l v e d . I t i s i n t e r e s t i n g to note that i n the case of microsomes from the southern armyworm midgut, the morphological appearance of the membrane v e s i c l e s prepared i n 0.25M sucrose and 0.15M KC1 a r e q u i t e d i s t i n c t ; thus although both f r a c t i o n s e x h i b i t e s s e n t i a l l y i d e n t i c a l l e v e l s of microsomal oxidase a c t i v i t y the v e s i c l e s prepared i n sucrose are r e l a t i v e l y smooth and rounded compared with the angular appearance of those prepared i n KC1. As a r e s u l t of the sometimes broad d i s t r i b u t i o n of microsomal oxidase a c t i v i t y i n s e v e r a l s u b c e l l u l a r f r a c t i o n s , the a c t i v i t y measured i n the microsomal f r a c t i o n per se may be a poor i n d i c a t o r of the t o t a l enzyme c a p a b i l i t y of a given i n s e c t t i s s u e . In comparative s t u d i e s , t h e r e f o r e , where a measure of t o t a l metabolic c a p a c i t y i s o f t e n r e q u i r e d , i t may be more appropriate to measure the a c t i v i t y i n l e s s homogeneous f r a c t i o n s such as the 10 OOOxg supernatant or even i n crude homogenates. Indeed, such preparat i o n s are probably most convenient i n many metabolic s t u d i e s s i n c e t h e i r use s i g n i f i c a n t l y decreases the time of p r e p a r a t i o n . The s t a b i l i t y of enzyme a c t i v i t y i n homogenates and s u b c e l l u l a r f r a c t i o n s from i n s e c t t i s s u e s v a r i e s considerably depending on the t i s s u e source, the method of p r e p a r a t i o n and the storage c o n d i t i o n s employed (8,9,10). As a general r u l e they should be used without delay a f t e r p r e p a r a t i o n . Oxidase a c t i v i t y i n preparations from whole house f l i e s or house f l y abdomens u s u a l l y i s r a p i d l y l o s t on storage at 0-2°C (8,10,31,43) but may be r e t a i n e d f o r s e v e r a l weeks under c e r t a i n c o n d i t i o n s (8,10,31). F r e e z i n g of these microsomes has been reported t o be d e l e t e r i o u s (8,10,51). However, microsomes from armyworm gut may be stored f o r up to a month as w e l l - d r a i n e d p e l l e t s at -15°C (59) (Table 5) and microsomal suspensions from c r i c k e t Malpighian tubules l o s t only about 15% of t h e i r i n i t i a l epoxidation a c t i v i t y a f t e r 3 weeks at the same temperature (38). 2

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270 TABLE 5 E f f e c t of f r e e z i n g on oxidase a c t i v i t y content

and cytochrome P-450

i n armyworm midgut microsomal preparations Oxidase a c t i v i t y

Storage (weeks) 0 1 2 3 4

3 (nmoles/mg protein/min) (xlO ) Epoxidation Hydroxylat i o n 92.5 92.5 88.0 94.6 98.2

Cytochrome P-450 (nmoles/mg p r o t e i n )

46.5 54.2 46.0 48.4

Data from K r i e g e r and Wilkinson (59) Microsomes were stored as w e l l - d r a i n e d p e l l e t s at

1.37 1.30 1.37 1.48

-15°C

Measurement of enzymatic a c t i v i t y Following p r e p a r a t i o n of a s u i t a b l e s u b c e l l u l a r f r a c t i o n with which to work the next step i s to measure i t s enzymatic a c t i v i t y towards an appropriate s u b s t r a t e . The i n c u b a t i o n mixture and c o n d i t i o n s to be used w i l l c l e a r l y depend on the nature of the enzyme, s u b c e l l u l a r f r a c t i o n and substrate under i n v e s t i g a t i o n and, of course, the purpose of the study. The o p t i m i z a t i o n of i n c u b a t i o n and assay c o n d i t i o n s i s extremely important and can i n i t s e l f provide a great d e a l of information on the nature and mechanism of the enzyme concerned. Unfortunately i t i s o f t e n overlooked or given minimal a t t e n t i o n where the s u b c e l l u l a r f r a c t i o n s are being used p r i m a r i l y as biochemical t o o l s to generate metabolites. The procedures employed to optimize c o n d i t i o n s f o r measuring enzyme r e a c t i o n s i n i n s e c t s u b c e l l u l a r f r a c t i o n s are e s s e n t i a l l y those p e r t a i n i n g to mammalian systems. Major parameters which should be evaluated are the type, s t r e n g t h and pH of the b u f f e r , the temperature under which the incubations are conducted and the a d d i t i o n of appropriate c o f a c t o r s or other m a t e r i a l s . Phosphate or T r i s b u f f e r s of v a r y i n g pH (7.0-8.5) and i o n i c s t r e n g t h u s u a l l y prove adequate f o r most i n v i t r o s t u d i e s on x e n o b i o t i c metabolism (8,9,10). Although most of the e a r l y i n v i t r o s t u d i e s were c a r r i e d out at 37°C i t i s now g e n e r a l l y agreed that i n s e c t enzymes seem to f u n c t i o n more s a t i s f a c t o r i l y at a temperature of about 30°C (8) and i n many cases optimum values of 20-25°C have been reported (35,36). Since enzyme a c t i v i t y i n many i n s e c t preparations i s l e s s s t a b l e than that i n corresponding mammalian f r a c t i o n s , l i n e a r i t y of the r e a c t i o n with respect to both time and p r o t e i n c o n c e n t r a t i o n should be a s c e r t a i n e d . The a d d i t i o n of c o f a c t o r s i s u s u a l l y necessary to o b t a i n maximal i n v i t r o a c t i v i t y and the nature of these c l e a r l y depends

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on the type of enzyme under study. For the enzymes i n v o l v e d i n x e n o b i o t i c metabolism there are only a few major types of cofactors. Microsomal o x i d a t i o n a c t i v i t y i s dependent p r i m a r i l y on the a d d i t i o n of NADPH or an appropriate generating system, the l a t t e r o f t e n proving p r e f e r a b l e by extending the time l i n e a r i t y of the r e a c t i o n (8). Incubations must be conducted a e r o b i c a l l y as oxygen i s a requirement f o r oxidase a c t i v i t y . Although m a t e r i a l s such as EDTA and/or nicotinamide are o f t e n added to enhance microsomal a c t i v i t y i n mammalian microsomes where they s t a b i l i z e NADPH through b l o c k i n g l i p i d p e r o x i d a t i o n and p y r i d i n e n u c l e o t i d a s e a c t i v i t y r e s p e c t i v e l y , t h e i r i n c l u s i o n i n i n s e c t microsomes appears to have l i t t l e or no b e n e f i c i a l e f f e c t on oxidase a c t i v i t y (8). T h i s i s a l s o g e n e r a l l y true of most metal ions (8) BSA (1-2% w/v) i s sometime act the a c t i o n s of r e s i d u a taken that t h i s or other a d d i t i o n s do not have other d e l e t e r i o u s e f f e c t s on enzyme a c t i v i t y . Soluble enzymes of importance i n x e n o b i o t i c metabolism i n c l u d e a v a r i e t y of hydrolases (phosphatases, carboxyesterases, amidases and p y r e t h r o i d hydrolases) (20), s e v e r a l g l u t a t h i o n e (GSH) r e q u i r i n g enzymes ( a l k y l and a r y l t r a n s f e r a s e s , DDTdehydrochlorinase and organothiocyanate metabolizing enzymes) (21,22) and numerous other conjugating enzymes ( g l u c o s y l t r a n s ­ f e r a s e , sulphotransferase, etc.) (22). The p r o p e r t i e s and i n v i t r o requirements, many of these enzymes have already been discussed i n t h i s symposium. Perhaps a few words should be added at t h i s point with respect to the substrate employed i n i n v i t r o s t u d i e s . Unfortu­ n a t e l y most of the substrates which are used i n x e n o b i o t i c s t u d i e s are of n e c e s s i t y h i g h l y l i p o p h i l i c m a t e r i a l s and t h e i r a d d i t i o n to an aqueous i n c u b a t i o n medium o f t e n presents a problem. U s u a l l y t h i s can be achieved by d i s s o l v i n g the substrate i n a small volume of ethanol, acetone or other organic solvent (care should be taken to ensure that t h i s does not i n h i b i t the enzyme) but even then i t can o f t e n be observed to p r e c i p i t a t e out of the i n c u b a t i o n medium. This r a i s e s s e r i o u s questions about the a c t u a l concentration of substrate i n the incubation mixture and how t h i s should be expressed. Studies with microsomal preparations from mammalian l i v e r and armyworm midgut have shown that i n the presence of a constant amount of microsomal p r o t e i n , the amount of a l d r i n epoxidized to d i e l d r i n i s independent of the t o t a l volume of the r e a c t i o n mixture and r e l a t e d only to the absolute amount of a l d r i n added (Table 6) (60,61). T h i s suggests that the true concentra­ t i o n of substrate a v a i l a b l e f o r enzyme conversion i s not that expressed by the molar concentration c a l c u l a t e d from the t o t a l incubation volume but i s probably the unknown concentration e x i s t i n g i n the l i p i d phase of the microsomes. There are, there­ f o r e , problems inherent i n expressing the concentrations of l i p o p h i l i c substrates i n molar terms and Κ values and other

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

272 TABLE 6

E f f e c t of a l d r i n s o l u b i l i t y on epoxidation i n p i g l i v e r microsomes Incubation Medium Total vol. (ml)

1 2 3 4

Microsomal suspension (ml)

Aldrin added

0.2 0.2 0.2 0.2

Dieldrin produced

(yg)

(yg)

50 50 50 50

9.2 9.3 9.4 10.2

Aldrin Concentration (yg/ml)

(μΜ)

50 25 12.5 10

137 68.,5 45..7 34..3

Data from Lewis et^ a l . a/ — Based on t o t a l volume of i n c u b a t i o n medium k i n e t i c parameters reported i n t h i s way should be accepted with r e s e r v a t i o n . Perhaps the best way of expressing substrate concen­ t r a t i o n i s i n terms of the absolute amount added to the r e a c t i o n medium s i n c e the a c t u a l c o n c e n t r a t i o n i s d i r e c t l y r e l a t e d to t h i s value. P a r t i c u l a r l y i n the case of the microsomal oxidase system, the choice of s u b s t r a t e s f o r i n v i t r o s t u d i e s i s l a r g e and many e x c e l l e n t and s e n s i t i v e assays are c u r r e n t l y a v a i l a b l e . Initial s t u d i e s to optimize the c o n d i t i o n s f o r some r e a c t i o n or to charac­ t e r i z e a given s u b c e l l u l a r f r a c t i o n are o f t e n f a c i l i t a t e d by using a model substrate which y i e l d s a s i n g l e metabolic product. Having thus e s t a b l i s h e d the c h a r a c t e r i s t i c s of the system these can u s u a l l y be a p p l i e d d i r e c t l y to s t u d i e s with more complex drug or i n s e c t i c i d e substrates which may have s e v e r a l s i t e s at which enzyme a t t a c k can occur and which consequently produce s e v e r a l d i f f e r e n t metabolites. A p p l i c a t i o n of i n v i t r o s t u d i e s To date, the major a p p l i c a t i o n of i n v i t r o s t u d i e s with i n s e c t s u b c e l l u l a r f r a c t i o n s has been to complement i n v i v o s t u d i e s on the metabolic f a t e and pathways of i n s e c t i c i d e chemi­ cals. In v i v o s t u d i e s are o f t e n complicated by the f a c t that the t e r m i n a l metabolic products (conjugates) are the ones commonly observed and these o f t e n provide l i t t l e information on the nature of primary or intermediary metabolites which may e x i s t at t r a c e l e v e l s or be of a t r a n s i e n t nature. In i n s e c t s the s i t u a t i o n i s compounded by the f a c t that with h i g h l y t o x i c m a t e r i a l s only very small doses of m a t e r i a l can be a p p l i e d without k i l l i n g the i n s e c t . With i n v i t r o systems t o x i c i t y does not have to be considered and high concentrations of t o x i c a n t s can be employed. Furthermore, the use of v a r i o u s s u b c e l l u l a r f r a c t i o n s permits the i n d i v i d u a l

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8.

WILKINSON

Insect Subcellufor

Components

273

study of i s o l a t e d components of the o v e r a l l metabolic machinery i n a r e l a t i v e l y concentrated form without the complications a r i s i n g from the presence of other metabolic components. Thus using a s u i t a b l y f o r t i f i e d microsomal f r a c t i o n the nature of the primary o x i d a t i v e metabolites can be studied i n the absence of the type I I conjugating systems found l a r g e l y i n the s o l u b l e f r a c t i o n . E a r l y i n v i v o metabolic s t u d i e s with the carbamates, f o r example, showed that these i n s e c t i c i d e s were r a p i d l y metabolized by i n s e c t s to water s o l u b l e products and many of which were conju­ gates of primary hydroxylated metabolites (5) (Figure 7). Subse­ quent i n v i t r o s t u d i e s u s i n g i s o l a t e d microsomal and s o l u b l e f r a c t i o n s have enabled these primary and secondary products to be more r e a d i l y i d e n t i f i e d and the appropriate enzyme systems b e t t e r characterized. Of p a r t i c u l a r s i g n i f i c a n c components i n i n v i t r o metabolis e x i s t s f o r manipulating the system by the omission or a d d i t i o n of s p e c i f i c c o f a c t o r s or i n h i b i t o r s . Thus the requirement of a c e r t a i n s u b c e l l u l a r f r a c t i o n f o r a p a r t i c u l a r c o f a c t o r to produce a c e r t a i n metabolite o f t e n provides a f a i r l y c l e a r p i c t u r e of the type of enzyme which i s i n v o l v e d i n the conversion. This type of experiment i s e s p e c i a l l y i n f o r m a t i v e where a compound may be metabolized to the same product by two d i f f e r e n t enzyme systems. The data shown i n Table 7 were obtained during the course of a study of d i a z i n o n and diazoxon metabolism i n house f l i e s (62). TABLE 7 E f f e c t of reduced g l u t a t h i o n e and NADPH on the degradation of d i a z i n o n and diazoxon by house f l y s u b c e l l u l a r f r a c t i o n s i n v i t r o Enzyme^ . source— Microsomes :

Soluble fraction:

Cofactors

Degradation Diazinon

None

0

GSH

3.2

ο (mymoles/hr/g + abdomen) Diazoxon 0 0

NADPH

122.1

23.2

NADPH + GSH

164.3

27.0

52.3

5.2

259.0

80.6

NADPH

47.0

1.5

NADPH + GSH

251.0

78.4

None GSH

Data from Yang _et a l . (62) a/ 9 — Microsomes and s o l u b l e f r a c t i o n s equivalent t o 35 and 10 + house f l y abdomens r e s p e c t i v e l y

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

274

In In Microsomal

vivo vitro Soluble

fraction

?

fraction

Cofactors

N A D P H , O-

OCNHCH^HMHCH OH

CH,S

2

Glucosides Sulfates .

( C H

3

) N ^

CH , Η Ν - * ^

V

^ C H

OCH

5

4OH PRIMARY

OXIDATIVE

SECONDARY CONJUGATES

METABOLITES

Figure 7.

EtO

Metabolism

of carbamate insecticides by microsomal and soluble fractions

EtO^ ^ O

x

Ρ Εt o '

Microsomes NADPH, 0

ν

—

θγ

Ν

γθΗ(ΟΗ ) 3

kyN CH a

2

Soluble GSH Microsomes NADPH,0 2

EtQ^ S(0) Ρ #

Eto' ^ O H "

Microsomes NADPH,0 2

+ unknowns Figure 8.

Metabolism of diazinon and diazoxon (62)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

:

8.

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These c l e a r l y show that because of the requirement f o r NADPH the microsomal degradation of d i a z i n o n and diazoxon i s p r i m a r i l y o x i d a t i v e i n nature. In c o n t r a s t the s o l u b l e f r a c t i o n contains an e f f i c i e n t GSH-dependent enzyme capable of degrading both compounds. The r e s u l t s of these s t u d i e s combined with an a n a l y s i s of the metabolites found i n the i n v i t r o incubations i n d i c a t e d that d i a z i n o n could be e i t h e r o x i d a t i v e l y a c t i v a t e d ( d e s u l f u r a ­ t i o n ) to diazoxon or could be degraded by microsomal o x i d a t i o n or s o l u b l e GSH-transferase to d i e t h y l phosphorothioic a c i d . Diazoxon was l i k e w i s e s u s c e p t i b l e to the two degradation pathways to y i e l d d i e t h y l phosphoric a c i d (Figure 8). Several groups of compounds are known to act as i n h i b i t o r s of the enzymes i n v o l v e d i n x e n o b i o t i c metabolism and some of these have value as i n s e c t i c i d e s y n e r g i s t s which enhance the i n s e c t i c i d a l potency of v a r i o u s m a t e r i a l microsomal o x i d a t i o n i n c l u d t i v e s , a r y l - 2 - p r o p y n y l e t h e r s , 1,2,3-benzothiadiazoles and a l a r g e number of s u b s t i t u t e d imidazoles (63). Many of the s o l u b l e hydrolases such as carboxylesterase and the p y r e t h r o i d metaboliz­ ing esterases are e f f e c t i v e l y blocked by phosphates such as t e t r a ethylpyrophosphate and t r i - o - c r e s y l phosphate (20,64) or carbamates such as 1-naphthyl N-propylcarbamate (65) and many GSHt r a n s f e r a s e s are blocked by compounds such as p-chloromercuribenzoate (21). L i k e the v a r i o u s c o f a c t o r s p r e v i o u s l y discussed such i n h i b i t o r s can be extremely u s e f u l i n p i n p o i n t i n g the enzymes involved i n c e r t a i n r e a c t i o n s . For example i n some cases a x e n o b i o t i c may be metabolized by more than one type of enzyme system i n the same s u b c e l l u l a r fraction. In t h i s case s p e c i f i c enzyme i n h i b i t o r s can be employed to d i s c r i m i n a t e between the two enzymes. To i l l u s t r a t e how t h i s technique can be a p p l i e d i n v i t r o we w i l l use the r e s u l t s from a mammalian study. I t has been known f o r some time that many of the modern p y r e t h r o i d s are metabolized i n mammals by both esterases and mixed-function oxidases i n l i v e r microsomes (66). Recent jLn v i t r o s t u d i e s have e s t a b l i s h e d s t r u c t u r e - b i o d e g r a d a b i l i t y r e l a t i o n s h i p s with a s e r i e s of 44 p y r e t h r o i d s and model compounds and have c l e a r l y shown the comparative r o l e of esterases and oxidases i n t h e i r metabolism. Each of the t e s t compounds was incubated with microsomes alone (esterase a c t i v i t y ) with microsomes + NADPH (esterase plus oxidase a c t i v i t y ) and with microsomes + NADPH + tetraethylpyrophosphate, an esterase i n h i b i t o r (oxidase a c t i v i t y ) . The r e s u l t s shown i n Table 8 f o r the isomers of phenothrin and cyanophenothrin t y p i f y the r e s u l t s obtained with a l l compounds t e s t e d . Thus the primary a l c o h o l e s t e r s of IR, t r a n s - s u b s t i t u t e d cyclopropane c a r b o x y l i c a c i d s (e.g. phenothrin) were most r a p i d l y metabolized by both esterase and oxidase a t t a c k , the corresponding IR, c i s isomers were degraded mainly by oxidase a c t i o n and were q u i t e r e s i s t a n t to esterase a t t a c k and the α-cyano compounds were r e s i s t a n t to the degradative a c t i o n of both types of enzymes.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

276 TABLE 8

E f f e c t of s t r u c t u r e on i n v i t r o metabolism of 3-phenoxybenzyl chrysanthemum e s t e r s by mouse l i v e r microsomes Structure

(IR, trans

isomer)

Percent metabolism r a t e r e l a t i v e

Compound and

isomer

Phenothrin (R = IR,

Esterase

Oxidase

+

Oxidase calc.

H)

trans

IR, çis Cyanophenothrin (R = IR,

t r a n s , a-RS

IR,

c i s , a-RS

59 + 3

27 + 2

78

+7

86

mouse; ( » , rabbit; (O), dog (14).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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c h l o f e n v i n p h o s i n these s p e c i e s i s 10 (rat), 100 (mouse), 500 (rabbit), a n d > 1200 (dog). T h u s the i n v i t r o e x p e r i m e n t s w o u l d have c o r r e c t l y p r e d i c t e d t h e o r d e r of acute t o x i c i t y i n the f o u r species. O n l y two i n v i t r o s t u d i e s c o u l d be found i n w h i c h the s a m e s p e c i e s a n d the s a m e d r u g s w e r e c o m p a r e d . T h e s e w e r e t h e r e p o r t s of C h h a b r a et a l . (15) w h o c o m p a r e d the m i c r o s o m a l o x i d a s e a c t i v i t i e s of l i v e r a n d i n t e s t i n e a g a i n s t f o u r c o m p o u n d s in f i v e s p e c i e s ( h a m s t e r , g u i n e a pig, r a t , m o u s e , r a b b i t ) a n d L i t t e r s t e t a l . (16) w h o u s e d s o m e of the s a m e d r u g s t o c o m p a r e m i c r o s o m a l m e t a b o l i s m i n lung, l i v e r , and k i d n e y of the s a m e s p e c i e s . T a b l e II shows that they found v e r y l i t t l e d i f f e r e n c e i n c y t o c h r o m e P-450 content h a v i n g h i g h e r P-450 l e v e l s t u d i e s a g r e e i n r a n k i n g t h e r a t as the l o w e s t of the f i v e s p e c i e s and i n the n a r r o w range, l e s s than 2 - f o l d , of c y t o c h r o m e P-450 concentration. T a b l e II.

A C o m p a r i s o n of H e p a t i c C y t o c h r o m e P-450 i n F i v e Species

L i t t e r s t et a i . (16) A A 490-450/mg Species protein /ml

C h h a b r a et a l . (15) n m o l e s /mg protein Species

rabbit hamster guinea p i g mouse rat

guinea pig hamster rabbit mouse rat

0. 177 0.140 0. 125 0. 108 0.098

+ Ο.Οβδ^ + 0.023 + 0.038 + 0.022 +0.025

1.45 Ι.26 1.1 1.1 0.84

b/ + 0. Ιό+ 0.07 +0.32 +0.07 + 0.07

- ± S. D. ~ ± S. E . T h e r e w a s r e a s o n a b l e a g r e e m e n t i n the r a n k i n g of the f i v e s p e c i e s a c c o r d i n g to t h e i r m i c r o s o m a l a n a l i n e h y d r o x y l a s e a c t i v i t i e s , T a b l e ΙΠ, the h a m s t e r a n d m o u s e b e i n g h i g h e s t i n b o t h s t u d i e s , a n d b o t h s h o w i n g the r a b b i t and g u i n e a pig a s the l o w e s t . T h e a c t i v i t i e s d i f f e r e d about 4 - f o l d i n the L i t t e r s t et a l . study. In the c a s e of b i p h e n y l h y d r o x y l a s e , T a b l e IV, the t w o s t u d i e s a g r e e o n the r a t a s l o w e s t i n l i v e r h y d r o x y l a s e a c t i v i t y but t h e r e i s l i t t l e a g r e e m e n t i n the r a n k i n g of the other s p e c i e s . The m e a n a c t i v i t i e s f o r the b i phenyl h y d r o x y l a s e v a r i e d about 3-fold i n both studies. F r o m this l i m i t e d r e v i e w of r e s u l t s f r o m two l a b o r a t o r i e s , it a p p e a r s that the technique i s r e a s o n a b l y r e l i a b l e i n the r a n k i n g

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T a b l e III. A C o m p a r i s o n of H e p a t i c A n a l i n e H y d r o x y l a s e i n F i v e Species L i t t e r s t et a l . (16) Activity Species

n m o l e s /mg protein/min

hamster mouse rat g u i n e a pig rabbit

2.7 + 0 . 9 ^ 1.5+0.3 0.8 + 0. 0.8 + 0. 0.6 + 0.4

C h h a b r a et a l . (15) Activity Species

n m o l e s /mg protein/15 m i n

mouse hamster

24.1 + 1 . 9 ^ 19.1 + 1.3

rabbit

9.8 + 2.3

- ± S. D.

T a b l e I V . A C o m p a r i s o n of H e p a t i c B i p h e n y l H y d r o x y l a s e i n F i v e Species L i t t e r st e t a l . (16) Activity Species guinea p i g hamster mouse rabbit rat

- ± S. D.

n m o l e s /mg protein/min 4.1 3.4 2.8 1.7 1.6

+ ± ± ± ±

3. 11.3 0.6 0.5 0.4

C h h a b r a et a l . (15) Activity Species mouse guinea pig rabbit hamster rat

n m o l e s /mg protein/15 m i n 95.3 + 1 0 . 0 ^ 7 7 . 9 + 1.8 5 8 . 1 + 1.6 4 7 . 4 + 3.0 34.4± 1.4

^ ± S . E .

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of m i c r o s o m a l o x i d a s e a c t i v i t y i f the d i f f e r e n c e s b e t w e e n s p e c i e s a r e at l e a s t 2 - f o l d . In c o m p a r i n g the e n z y m e a c t i v i t i e s of T a b l e III and I V w i t h the c y t o c h r o m e P-450 c o n c e n t r a t i o n s s h o w n i n T a b l e II, i t w i l l be s e e n that t h e r e i s often l i t t l e c o r r e l a t i o n b e t w e e n the two p a r a m e t e r s e v e n though c y t o c h r o m e P-450 i s k n o w n to be the k e y e n z y m e i n these o x i d a s e s y s t e m s . T h i s h a s a l s o b e e n o b s e r v e d i n s t u d i e s of s i n g l e s p e c i e s . Indeed, i n v i e w of o u r p r e s e n t u n d e r s t a n d i n g of this i m p o r t a n t h e m o p r o t e i n , a d i r e c t r e l a t i o n s h i p o n the b a s i s of c y t o c h r o m e P-450 content a l o n e w o u l d b e s u r p r i s i n g s i n c e t h i s e n z y m e i s k n o w n to e x i s t i n s e v e r a l f o r m s with different substrate specificities. A c a r e f u l study o i n v o l v e d i n the a c t i v a t i o s p e c i e s f a i l e d to p r o v i d e a n e x p l a n a t i o n f o r the d i f f e r i n g t o x i c i t i e s of p a r a t h i o n (17). T h i s i n s p i t e of the f a c t that t h e r e w e r e l a r g e d i f f e r e n c e s , 2 0 - f o l d i n p a r a o x o n f o r m a t i o n and 8 - f o l d i n p a r a o x o n d e g r a d a t i o n , a m o n g the s p e c i e s s t u d i e d . T h e a u t h o r s c o n c l u d e d that o r g a n s o r t i s s u e s other than the l i v e r m u s t b e i n v o l v e d i n p r o t e c t i n g the a n i m a l a g a i n s t s u c h t o x i c a n t s . O f c o u r s e i t i s a l s o p o s s i b l e that the t a r g e t of the t o x i c a n t , the a c e t y l c h o l i n e s t e r a s e s y s t e m , a l s o v a r i e d i n s e n s i t i v i t y among the nine species. T h e a g r e e m e n t b e t w e e n i n v i t r o m e t a b o l i s m and t o x i c i t y i n f i v e m a m m a l i a n and t h r e e a v i a n s p e c i e s w a s a l s o u n s a t i s f a c t o r y i n a study of the i n s e c t i c i d e , d i a z i n o n (18). O n l y w i t h the sheep, w h i c h c a n t o l e r a t e 2-50 t i m e s m o r e d i a z i n o n t h a n pigs, g u i n e a pigs, cows, r a t s , t u r k e y s , c h i c k e n s , and d u c k s , w a s the t o x i c i t y relatively w e l l c o r r e l a t e d w i t h i n vitro m e t a b o l i s m . The authors c o n c l u d e d that e x t r a h e p a t i c m e t a b o l i s m w a s m o r e i m p o r t a n t than l i v e r m e t a b o l i s m . F a c t o r s A f f e c t i n g C o m p a r i s o n s - - S o u r c e s of E r r o r T h e r e a r e s e v e r a l b i o l o g i c a l , experimental, and even e n v i r o n m e n t a l f a c t o r s w h i c h c a n a f f e c t the p e r f o r m a n c e of i n v i t r o s y s t e m s . S o m e e x a m p l e s w h i c h a r e to b e found i n the c u r rent l i t e r a t u r e include: 1. S e x and age e f f e c t s : D e W a i d e (19) found that the v a r i a t i o n i n a m i n o p y r i n e N - d e m e t h y l a s e and a n i l i n e h y d r o x y l a s e a c t i v i t y b e t w e e n i n d i v i d u a l s i n four s p e c i e s of f i s h o b s c u r e d a n y p o s s i b l e s e x d i f f e r e n c e s i n the a c t i v i t i e s of t h e s e e n z y m e s . S e x d i f f e r e n c e s i n m i c r o s o m a l o x i d a s e a c t i v i t y a r e w e l l known, h o w e v e r . F o r e x a m p l e , the r a t and m o u s e e x h i b i t s e x

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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d i f f e r e n c e s i n k i n e t i c c o n s t a n t s ( K and V ) but the g u i n e a pig, r a b b i t , and m o n k e y do not (20). A n o t h e r e x a m p l e i s s e e n i n the r e p o r t of W h i t e h o u s e et a l . (17) who found a s e x d i f f e r e n c e i n the d e s u l f u r a t i o n of p a r a t h i o n i n two of nine s p e c i e s t e s t e d , guinea pig and r a t , and i n i t s h y d r o l y s i s i n o n l y one, the r a t . D i f f e r e n c e s i n the r a t e of e n z y m e d e v e l o p m e n t a r e w e l l k n o w n f r o m s t u d i e s of the c o m m o n l a b o r a t o r y a n i m a l s , h e n c e s i m i l a r c h a r a c t e r i s t i c s s h o u l d be e x p e c t e d i n other s p e c i e s . Some i n v e s t i g a t o r s f a i l to take t h i s into account i n c o m p a r i s o n s of i n v e r t e b r a t e s o r of w i l d v e r t e b r a t e s , h o w e v e r , p e r h a p s b e c a u s e they have no c h o i c e o r b e c a u s e they a r e u n a w a r e of the p o s s i b l e effects of age d i f f e r e n c e s . E x t r e m e v a r i a t i o n s i n m i c r o s o m a l oxidase activit stages of d e v e l o p m e n t a ( F i g u r e s 6, 7, 8) show. S i m i l a r o b s e r v a t i o n s have b e e n m a d e w i t h the e s t e r a s e s w h i c h m e t a b o l i z e j u v e n i l e h o r m o n e a n a l o g s (21, 22) and w i t h e p o x i d e h y d r a s e w h i c h m e t a b o l i z e s the j u v e n i l e h o r m o n e (23). A g e dependent f l u c t u a t i o n s i n m i c r o s o m a l o x i d a s e a c t i v i t y have a l s o b e e n s e e n i n the h o u s e c r i c k e t (24) and the c o c k r o a c h (25). S p e c i e s d i f f e r e n c e s i n the r a t e of d e v e l o p m e n t have b e e n o b s e r v e d w i t h the g l u c u r o n i d a t i o n s y s t e m (26). C h i c k e m b r y o s w e r e a l m o s t as a c t i v e i n the g l u c u r o n i d a t i o n of O - a m i n o p h e n o l at 12 d a y s as i n 6 w e e k - o l d c o c k r e l s w h e r e a s the l i v e r of the 16 d a y - o l d m o u s e fetus was n e g a t i v e and e v e n the 10-day i n f a n t l i v e r c o n t a i n e d o n l y 5 0 % of the a d u l t a c t i v i t y . 2. S o u r c e of e n z y m e s ; D e W a i d e (19) m e a s u r e d the a p p a r e n t V and K f o r the _p_-hydroxylation of a n i l i n e of e i g h t t i s sues, l i v e r , kidney, h e a r t , lung, gut, m u s c l e , blood, and s p l e e n i n pigeon, r a t , trout, r o a c h , and c r a b ( s u b s t i t u t i n g hepatopanc r e a s f o r l i v e r i n the l a t t e r ) . In the c r a b , t r o u t , and r o a c h , he a l s o i n c l u d e d the g i l l i n t h e s e m e a s u r e m e n t s . The l i v e r was the m o s t a c t i v e s o u r c e of e n z y m e i n the f o u r v e r t e b r a t e s , but the c r a b g i l l was c o n s i d e r a b l y m o r e a c t i v e t h a n i t s h e p a t o p a n c r e a s . The k i d n e y was n e a r l y as a c t i v e as the l i v e r i n the c a s e of the t r o u t and b o t h the k i d n e y and lung w e r e i m p o r t a n t s o u r c e s of the e n z y m e i n the pigeon. P o h l et a l . (27) a l s o found d i f f e r e n c e s i n the d i s t r i b u t i o n of e n z y m e a c t i v i t y a m o n g the o r g a n s of d i f f e r e n t s p e c i e s . In the l i t t l e s k a t e , f o r e x a m p l e , the l i v e r and k i d n e y had a p p r o x i m a t e l y the s a m e a c t i v i t y , per mg of m i c r o s o m a l p r o t e i n , w h i l e i n the w i n t e r f l o u n d e r the l i v e r w a s a p p r o x i m a t e l y 20 t i m e s as a c t i v e as the k i d n e y . m

m

a

x

m

a

x

m

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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"Ό

Egg I

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Larva

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| Puparium |

299

Adult

"5

DAYS Figure 6.

Microsomal aldrin epoxidases activity in the developmental stages of Isohn-B and SR house flies

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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DEVELOPMENTAL STAGS (DAIS)

Pesticide Biochemistry and Physiology Figure 7.

O

Microsomal aldrin epoxidase activity in developmental stages of the blow fly (39)

U LARVA

1 8

1216 PUPARIUM DEVELOPMENTAL STAGE

2k

20 I

28

32

ADULT

(DAYS)

Pesticide Biochemistry and Physiology Figure 8.

Microsomal aldrin epoxidase activity in developmental stages of the fleshfly(39)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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T h e r e i s c o n s i d e r a b l e u n c e r t a i n t y about the s i t e of m f o a c t i v i t y i n i n v e r t e b r a t e a n i m a l s . P o h l et a l . (27) w e r e u n a b l e to d e t e c t h y d r o x y l a s e o r d e a l k y l a s e a c t i v i t y i n the h e p a t o p a n c r e a s of the l o b s t e r o r c r a b w h i l e B u r n s (28) d i d f i n d s o m e d r u g m e t a b o l i z i n g a c t i v i t y i n t h i s t i s s u e i n the c r a b but found m o r e i n g i l l and i n c l a w m u s c l e t i s s u e . D e W a i d e (19) a l s o found m o r e a c t i v i t y i n the g i l l t i s s u e of the c r a b t h a n i n the h e p a t o p a n c r e a s . E l m a m l o u k and G e s s n e r (29) w e r e a l s o u n a b l e to d e t e c t h y d r o x y l a s e o r d e m e t h y l a s e a c t i v i t y i n the m i c r o s o m a l f r a c t i o n of the h e p a t o p a n c r e a s of the l o b s t e r but t h e y d i d f i n d s o m e a n i l i n e h y d r o x y l a s e and n i t r o r e d u c t a s e a c t i v i t y i n the s o l u b l e fraction. A n interesting resul activity i n various tissue ity of c l a w m u s c l e . In t e r m s of s p e c i f i c a c t i v i t y , the g i l l m i c r o s o m e s w e r e c o n s i d e r a b l y m o r e a c t i v e than t h o s e of hepatopanc r e a s o r c l a w m u s c l e but the t o t a l c a p a c i t y of the m u s c l e s y s t e m was h i g h e r than that of g i l l and h e p a t o p a n c r e a s c o m b i n e d , 3.6 n m o l e s per h o u r c o m p a r e d to 1.3 a n d 1.2 n m o l e s per h o u r f o r g i l l and h e p a t o p a n c r e a s . 3. C e l l f r a c t i o n ; I n c o m p a r i n g the u s u a l c e n t r i f u g a l f r a c tions of l i v e r h o m o g e n a t e s of the r a t and the t r o u t , D e W a i d e (19) found that t r o u t m i c r o s o m e s c o n t a i n e d o n l y 4 7 % of the N_d e m e t h y l a s e a c t i v i t y of the homogenate w h e r e a s the r a t l i v e r m i c r o s o m e s c o n t a i n e d 7 9 % of the a c t i v i t y . E v e n the p r o c e s s of c e n t r i f u g i n g the 9000 x G f r a c t i o n (containing m i c r o s o m e s a n d s o l u b l e f r a c t i o n ) a t 100, 000 x G f o r 1 hour, then r e m i x i n g w i t h a t i s s u e g r i n d e r , r e s u l t e d i n a 3 1 % l o s s of a c t i v i t y b y the t r o u t s y s t e m c o m p a r e d to a n e g l i g i b l e l o s s b y the r a t s y s t e m . T h e s e r e s u l t s l e d D e W a i d e to use the 9000 x G f r a c t i o n i n h i s s p e c i e s c o m p a r i s o n s . A s he points out, t h i s not o n l y r e d u c e s e r r o r s due to the d i f f e r e n t i a l l o s s of a c t i v i t y i n t e s t s i n v o l v i n g d i f f e r e n t s p e c i e s and t i s s u e s , but i t s i m p l i f i e s the p r o c e d u r e s b y s h o r t e n ing the t i m e i n v o l v e d and e l i m i n a t i n g o t h e r o p p o r t u n i t i e s f o r l o s s of a c t i v i t y . O t h e r o b s e r v a t i o n s s u p p o r t the use of the m o r e i n c l u s i v e i n t e r m e d i a t e f r a c t i o n of t i s s u e h o m o g e n a t e s (e.g. the 9000 x G f r a c t i o n , e t c . ) . B u h l e r and R a s m u s s e n (30) found that the n i t r o r e d u c t a s e a c t i v i t y of s e v e r a l s p e c i e s of f i s h was a l m o s t e n t i r e l y i n the s o l u b l e f r a c t i o n w h i l e i n m a m m a l i a n s p e c i e s it i s e q u a l l y d i s t r i b u t e d b e t w e e n the m i c r o s o m a l and s o l u b l e f r a c t i o n (31 ). O t h e r e x a m p l e s of t h i s k i n d a r e the r e p o r t by E l m a m l o u k and G e s s n e r (29) w h o o b s e r v e d that the a n i l i n e h y d r o x y l a s e a c t i v i t y

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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of l o b s t e r h e p a t o p a n c r e a s h o m o g e n a t e s w a s i n the s o l u b l e f r a c t i o n w h i l e n i t r o r e d u c t a s e a c t i v i t y was i n b o t h m i c r o s o m a l and soluble fraction. E i g h t s p e c i e s w e r e s t u d i e d i n a d i r e c t c o m p a r i s o n of m i c r o s o m a l and w h o l e h o m o g e n a t e f r a c t i o n s of the l i v e r as the e n z y m e s o u r c e f o r the d e s u l f u r a t i o n of p a r a t h i o n (17). The two s y s t e m s a g r e e d i n the r e l a t i v e p o s i t i o n s of the f i r s t f i v e s p e c i e s but d i s a g r e e d i n the p o s i t i o n s of the l a s t t h r e e . The d i f f e r e n c e s a m o n g these t h r e e w e r e o n l y about 2 - f o l d , h o w e v e r . T h e i n s e c t e s t e r a s e s w h i c h h y d r o l y z e the j u v e n i l e h o r m o n e a n a l o g s a r e d i s t r i b u t e d b e t w e e n s o l u b l e f r a c t i o n (20%) and m i c r o s o m a l f r a c t i o n (40%) i n the h o u s e f l y (21) and f l e s h f l y (22) w h i l e the d i s t r i b u t i o n i n the c a s (60%) and m i c r o s o m a l f r a c t i o 4. E n z y m e s t a b i l i t y ; In c o m p a r i s o n s b e t w e e n the r a t and the r o a c h D e W a i d e (19) s u b j e c t e d the 9000 x G s u p e r n a t a n t f r a c t i o n to v a r i o u s t r e a t m e n t s p r i o r to i n c u b a t i o n w i t h the s u b s t r a t e . W h e n the e n z y m e s w e r e a l l o w e d to s t a n d f o r 5 h r s at r o o m t e m p e r a t u r e p r i o r to a s s a y the N - d e m e t h y l a t i n g s y s t e m of the r o a c h l o s t 4 2 % of i t s a c t i v i t y w h i l e that of the r a t l o s t 1 4 % . S i m i l a r t r e a t m e n t of the w h o l e h o m o g e n a t e (used i n the a s s a y of j D - n i t r o p h e n o l g l u c u r o n i d a s e ) r e s u l t e d i n a 6 2 % i n c r e a s e i n the a c t i v i t y of the r o a c h and a 1 6 % i n c r e a s e i n t h a t of the r a t . O t h e r t r e a t m e n t s ( s t o r a g e of the e n z y m e s at -15°C f o r 15 h r s , r e p e a t e d f r e e z i n g and t h a w i n g of the e n z y m e s , and u l t r a s o n i c a tion) a f f e c t e d the N - d e m e t h y l a s e and h y d r o x y l a s e s y s t e m s of the two s p e c i e s to a p p r o x i m a t e l y the s a m e extent. A d i f f e r e n c e i n e n z y m e s t a b i l i t y was a l s o o b s e r v e d i n a study of the e f f e c t of t e m p e r a t u r e on m i c r o s o m a l a l d r i n e p o x i d a s e i n e i g h t s t r a i n s of the house f l y (32). M i c r o s o m a l o x i d a s e s of the W o r l d H e a l t h O r g a n i z a t i o n S t a n d a r d R e f e r e n c e S t r a i n of f l i e s c o u l d be i n c u b a t e d at 47. 5°C f o r 5 m i n and s t i l l r e t a i n 8 1 % of t h e i r m a x i m u m e p o x i d a s e a c t i v i t y w h e r e a s s e v e n o t h e r s t r a i n s l o s t 6 2 - 9 0 % of t h e i r m a x i m u m a c t i v i t y a f t e r s u c h t r e a t m e n t . 5. T e m p e r a t u r e of i n c u b a t i o n ; The e r r o r w h i c h c a n o c c u r by the use of the w r o n g t e m p e r a t u r e i n i n c u b a t i o n s of m i c r o s o m a l o x i d a s e e n z y m e s c a n be a p p r e c i a t e d by a study of DeWaide's r e s u l t s i n w h i c h the t e m p e r a t u r e - a c t i v i t y c u r v e s w e r e d e t e r m i n e d f o r eight s p e c i e s , F i g u r e 9. In a l l c a s e s the r e g i o n of m a x i m u m a c t i v i t y w a s n a r r o w , about 5°C, d r o p p i n g off s h a r p l y at t e m p e r a t u r e s o u t s i d e t h i s r a n g e . D e W a i d e c o n c l u d e d , i n a g r e e m e n t w i t h e a r l i e r s t u d i e s b y A d a m s o n (6) and o t h e r s , that the l i v e r d e m e t h y l a s e of b i r d s (based on s t u d i e s w i t h the pigeon) s h o u l d be i n c u b a t e d at 42 C., t h o s e of m a m m a l s at 37 C and

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activity

e

temperature ( C )

temparaturef

C)

J. H. DeWaide (thesis) Figure 9. Ν-demethylation of aminopyrine by 9000 g supernatants prepared from liver homogenates of various animal species. Activity was measured for 10 min at different temperatures. The values of each species were expressed in relation to the maximal value (numerically 100) (19).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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304

METABOLISM

t h o s e of f i s h at 25°C. T h e s e t e m p e r a t u r e s w o u l d not be o p t i m u m i n e v e r y c a s e , h o w e v e r , as was s h o w n by f u r t h e r c o m p a r i s o n of e n z y m e a c t i v i t i e s at t h e s e and at the s p e c i f i c t e m p e r a t u r e o p t i m a for e a c h s p e c i e s . W i t h s o m e s p e c i e s the N - d e m e t h y l a s e a c t i v i t y was as l i t t l e as 4 6 % that o b s e r v e d at the s p e c i f i c o p t i m u m . The d i f f e r e n c e s w e r e l e s s i n the J D - h y d r o x y l a t i o n of a n i l i n e w h e r e the o p e r a t i v e t e m p e r a t u r e r e s u l t e d i n a c t i v i t i e s a t l e a s t 6 4 % of t h o s e o b t a i n e d i n the o p t i m u m r a n g e . P r e s u m a b l y i t was not p r a c t i c a l to c o n d u c t the i n c u b a t i o n s f o r e a c h s p e c i e s a t its o p t i m u m l e v e l . A n o t h e r e x a m p l e of a t e m p e r a t u r e effect d u r i n g e n z y m e a s s a y i s m e n t i o n e d b y B e n d (33). T h e l i v e r O - d e a l k y l a s e s y s t e m of the l i t t l e s k a t e was m u c h l e s s a c t i v e at b o t h 41 C and 12 C t h a n a t 30 °C. Recent reports indicat s u c h s t u d i e s i s s t i l l open. P o h l et a l . (27) i n c u b a t e d m i c r o s o m e s p r e p a r e d f r o m the l i v e r s of m a r i n e s p e c i e s o r the h e p a t o p a n c r e a s of c r a b and l o b s t e r at 30 C., w h i l e B u r n s (28) u s e d t e m p e r a t u r e s of 14.5 C and 20 C f o r m i c r o s o m e s f r o m the f i d d l e r c r a b and E l m a m l o u k and G e s s n e r (29, 34) i n c u b a t e d l o b s t e r he pato panc r e a s m i c r o s o m e s at 20°C. None of t h e s e a u t h o r s p r o v i d e d a t a i n s u p p o r t of t h e i r u s e of t h e s e t e m p e r a t u r e s . D e W a i d e (19) a l s o found a t e m p e r a t u r e d i f f e r e n t i a l i n the s t a b i l i t y of the N - d e m e t h y l a s e s y s t e m of r a t and t r o u t , the t r o u t s y s t e m r e m a i n i n g l i n e a r f o r a l o n g e r p e r i o d (20-30 m i n ) at i n c u b a t i o n t e m p e r a t u r e s n e a r i t s o p t i m u m , than that of the r a t (5-10 m i n ) w h e n i n c u b a t e d at i t s o p t i m u m . 6. K i n e t i c s ; A s i d e f r o m its v a l u e i n c h a r a c t e r i z i n g e n z y m e s , the k i n e t i c c o n s t a n t K i s u s e f u l i n the d e t e r m i n a t i o n of the o p t i m u m l e v e l of s u b s t r a t e to be u s e d i n m e a s u r i n g e n z y m e activity. A s a general practice substrate concentrations should be at l e a s t t w i c e the K to a c h i e v e m a x i m u m a c t i v i t y of the enzyme being measured. F r e q u e n t l y investigators comparing a c t i v i t y of a n e n z y m e i n d i f f e r e n t s p e c i e s use the s a m e s u b s t r a t e c o n c e n t r a t i o n , a p p a r e n t l y a s s u m i n g that the K f o r the e n z y m e i s the s a m e i n a l l s p e c i e s o r that the s u b s t r a t e c o n c e n t r a t i o n c h o s e n w i l l e x c e e d e v e n the h i g h e s t K . DeWaide's study ( T a b l e V) shows that t h i s a s s u m p t i o n i s not a l w a y s j u s t i f i e d . The K v a l u e s f o r N - d e m e t h y l a s e v a r i e d 2 4 - f o l d a m o n g the 16 s p e c i e s i n v e s t i g a t e d ( f r o m 0.42 m M f o r the p i g e o n to 10 m M f o r the pike) and 2 0 0 - f o l d f o r the h y d r o x y l a s e s y s t e m (0.05 m M f o r the r a t to 10 m M f o r the w h i t e b r e a m ) . The f o r the g l u c u r o n i d a s e s y s t e m , c o m p a r e d i n 11 s p e c i e s , v a r i e d m u c h l e s s , f r o m 0.32 m M f o r the m o u s e and l i z a r d to 1. 3 m M f o r the e e l . m

m

m

m

m

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

7.6 9.3 6.5 2.8 3.5 10 8.0

0.8 1.3 0.65 0.6 0.42 3. 23 3.0 6. 1 1.5

+ 3.5

+ 0.5 + 0.9

+ 2.7 + 0.6

+ 0.7

+ 0.07

+ 0.5 +0.20

N-demethylation of a m i n o p y r i n e ^

1.31 + 0.34 1.67 2.4 +0.3 4.5 4.2 5.3 +1.3 10 2.4 +0.4 2.6 0.48 + 0 . 1 2 1.7 +0.3 4.0 5.5 +2.0 0.3

0.8 0 . 2 9 +0.04 0.05 + 0 . 0 2

jp- H y d r o x y l a t i o n of aniline^-'

0.48 1.3 0.67

0. 97

0.47 0.83 0.53

0.64 0.33

0.32 0.72

± 0. 3

± 0. 14

+ 0-. 10

Gludur onidation of ^ - n i t r ο p h e n o l —

m

m

— K - v a l u e s i n mM; m e a n s of 2 o r m o r e d e t e r m i n a t i o n s ; f o r a n i m a l s of w h i c h the a p p a r e n t K - v a l u e s a r e d e t e r m i n e d by 4-10 d e t e r m i n a t i o n s t h e ^ t a n d a r d e r r o r of .the m e a n i s g i v e n . — A s s a y e d w i t h 9000 g —/Range of a m i n o p y r i n e c o n c e n t r a t i o n used: 1.5-35 m M . s u p e r n a t a n t of g i l l ^ R a n g e of a n i l i n e c o n c e n t r a t i o n used: 0. 1-22 m M . homogenate. — R a n g e of p_-nitrophenol c o n c e n t r a t i o n used: 0. 14-1.4 m M . D a t a f r o m D e W a i d e (19)*

m

A p p a r e n t K - V a l u e s of S u b s t r a t e s f o r H e p a t i c E n z y m e s . — '

hamster mouse rat hen pigeon lizard frog bream carp tench white b r e a m roach rudd rainbow trout eel pike sea l a m p r e y wool-handed c r a b —

T a b l e V.

XENOBIOTIC METABOLISM

306

K n o w l e d g e of a p p a r e n t K m and V x values is important i n a n o t h e r way, the i n t e r p r e t a t i o n of i n v i t r o d a t a i n t e r m s of the i n v i v o c o n d i t i o n s . T h i s was d i s c u s s e d by C a s t r o and G i l l e t t e (20) i n t h e i r s t u d i e s of the N - d e m e t h y l a t i o n of e t h y l m o r p h i n e b y m i c r o s o m e s of r a t , m o u s e , r a b b i t , g u i n e a pig, and m o n k e y . A s t h e y state, the d i f f e r e n c e s i n s u b s t r a t e c o n c e n t r a t i o n s b e t w e e n the i n v i t r o and i n v i v o s i t u a t i o n c o u l d o b s c u r e a d i f f e r e n c e i n e n z y m e a c t i v i t y i n one c a s e o r the o t h e r i f the K i s not t a k e n into a c c o u n t . 7. N a t u r a l i n h i b i t o r s of e n z y m e a c t i v i t y : S e v e r a l of the e a r l y f a i l u r e s to d e t e c t e n z y m e a c t i v i t y i n the t i s s u e s of i n s e c t s w e r e l a t e r found to be due to the p r e s e n c e of n a t u r a l i n h i b i t o r s w h i c h totally or p a r t i a l l s u r p r i s i n g s i n c e the s m a l of s p e c i f i c o r g a n s o r t i s s u e s . T h u s the h o m o g e n i z i n g of w h o l e i n s e c t s o r m a j o r body s e g m e n t s m i g h t be e x p e c t e d to i n t r o d u c e c a t a b o l i c e n z y m e s into the s u b c e l l u l a r f r a c t i o n b e i n g a s s a y e d . T h i s was the r e a s o n f o r low m f o a c t i v i t y i n l e p i d o p t e r o u s l a r v a e (35), the h o u s e c r i c k e t (36), and the honey bee (37). The i n h i b i t i o n of m f o a c t i v i t y i n m i c r o s o m e s p r e p a r e d f r o m w h o l e house f l i e s was t r a c e d to eye pigments (38) a n d was c o r ­ r e c t e d by r e m o v i n g the heads b e f o r e h o m o g e n i z a t i o n . S u c h i n h i b i t o r s a r e a l s o p r e s e n t i n the f l e s h f l y and b l o w f l y (39). Two n a t u r a l i n h i b i t o r s of h o u s e f l y m i c r o s o m a l o x i d a s e s have b e e n c h a r a c t e r i z e d by J o r d a n and S m i t h (40), one of t h e s e p r o b ­ a b l y the eye p i g m e n t i d e n t i f i e d by S c h o n b r o d and T e r r i e r e (38). A n o t h e r p o t e n t i a l s o u r c e of i n t e r f e r e n c e w i t h m i c r o s o m a l o x i d a s e a c t i v i t y i n s p e c i e s c o m p a r i s o n s i s the l i p i d p e r o x i d a s e s y s t e m . A 1 5 - f o l d d i f f e r e n c e i n the a c t i v i t y of t h i s s y s t e m b e t w e e n r a t l i v e r and r a b b i t l i v e r m i c r o s o m e s has b e e n r e p o r t e d (41). T h i s was s u f f i c i e n t to change the r e l a t i v e a c t i v i t y of the r a t d e m e t h y l a s e c o m p a r e d to that of the r a b b i t f r o m 2.2 t i m e s (rat m o r e than r a b b i t ) to 3.4 t i m e s w h e n the p e r o x i d a t i o n was p r e v e n t e d by the a d d i t i o n of E D T A . P o h l et a l . (27) c o u l d not d e t e c t m f o a c t i v i t y i n the hepatop a n c r e a s of l o b s t e r o r c r a b , a t t r i b u t i n g t h i s i n p a r t to the p r e s e n c e of d i g e s t i v e j u i c e s r e l e a s e d d u r i n g h o m o g e n i z a t i o n . T h e s e j u i c e s w e r e found to be i n h i b i t o r y of the h y d r o x y l a t i o n of a n i l i n e and the N_-demethylation of b e n z p h e t a m i n e . However, the i n h i b i t o r had o n l y a s l i g h t effect on the d e - e t h y l a t i o n of 7 - e t h o x y c ο uma r i η. T h i s o b s e r v a t i o n c o u l d e x p l a i n the f a i l u r e of other w o r k e r s (28, 29) to d e t e c t m i c r o s o m a l o x i d a s e a c t i v i t y i n the he pato p a n c r e a s of the c r a b and l o b s t e r . m

a

m

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

TERRiERE

Comparative

Xenobiotic

Metabolism

307

8. E n v i r o n m e n t a l e f f e c t s : D u r i n g h i s study of the d r u g m e t a b o l i z i n g a c t i v i t y of l i v e r m i c r o s o m e s f r o m f i s h s p e c i e s o b t a i n e d f r o m the R h i n e R i v e r a n d i t s t r i b u t a r i e s , D e W a i d e (19, 42) o b s e r v e d a s e a s o n a l v a r i a t i o n i n e n z y m e a c t i v i t y . F u r t h e r i n v e s t i g a t i o n r e v e a l e d that the N- de m e thy l a s e a c t i v i t y of the r o a c h ( F i g u r e 10) a n d the r u d d ( T a b l e V I ) c o l l e c t e d d u r i n g the s u m m e r m o n t h s w a s m o r e than t w i c e that of s p e c i m e n s c o l l e c t e d i n the w i n t e r . T h e d i f f e r e n c e w a s not e x p l a i n e d on the b a s i s of l i v e r w e i g h t o r p r o t e i n content or o n d i f f e r e n c e s i n w a t e r t e m ­ p e r a t u r e . It w a s c o n c l u d e d that the p r e s e n c e of e n z y m e i n d u c i n g c h e m i c a l s i n the f i s h e s e n v i r o n m e n t d u r i n g the s u m m e r m o n t h s was the m a i n c a u s e of t h e s e d i f f e r e n c e s i n e n z y m e a c t i v i t y . T h e r e i s now c o n s i d e r a b l species are induced b P C B ' s (43, 44, 45). I n c r e a s e s i n a r y l h y d r o c a r b o n h y d r o x y l a s e a c t i v i t i e s (benzo-(a )-pyrene as s u b s t r a t e ) up to 4 - f o l d have b e e n noted (46). H o w e v e r , not a l l s p e c i e s a r e a f f e c t e d nor a r e the e f f e c t s on the x e n o b i o t i c m e t a b o l i z i n g e n z y m e s the s a m e as noted by P a y n e (47). F o r e x a m p l e , the a r y l h y d r o c a r b o n h y d r o x y l a s e was i n d u c e d i n the t r o u t but N - d e m e t h y l a s e w a s not a f f e c t e d . N e i t h e r e n z y m e w a s i n d u c e d i n the c r a b a n d l o b s t e r . Y a w e t z (48) a l s o r a i s e d t h i s q u e s t i o n o n f i n d i n g b o t h D D E and P C B ' s i n the t i s s u e s of s i x s p e c i e s of b i r d s i n the i n v i t r o study of a l d r i n e p o x i d a t i o n . R e s i d u e c o n c e n t r a t i o n i n h e a r t t i s s u e ( l i v e r w a s not a n a l y z e d ) w e r e 0. 1-1.2 p p m f o r the t w o inducers. T h e r e m a y a l s o be a n e f f e c t o n e n z y m e a c t i v i t y b y the e n v i r o n m e n t a l t e m p e r a t u r e of the s p e c i e s under study. D e W a i d e (49) found that t r o u t and r o a c h h e l d a t 5 C f o r 2 w e e k s had l i v e r h y d r o x y l a s e and Ν - de m e thy las e a c t i v i t i e s about t w i c e those of f i s h h e l d at 18 C. In s i m i l a r e x p e r i m e n t s w i t h the h a m s t e r a n d the r a t ( t e m p e r a t u r e s 5 C a n d 23 C ) the d i f f e r e n c e s w e r e about 1. 5 - f o l d o r l e s s . 9* B a s i s of c o m p a r i s o n ; N e a r l y a l l i n v e s t i g a t o r s of x e n o ­ b i o t i c m e t a b o l i s m r e p o r t m e t a b o l i c a c t i v i t y i n t e r m s of the p r o ­ t e i n content of the f r a c t i o n b e i n g a s s a y e d . T h i s i s b a s e d o n the a s s u m p t i o n that the p r e p a r a t i v e m e t h o d s u s e d w i l l r e s u l t i n c o n ­ stant and r e p r o d u c i b l e a m o u n t s of e n z y m e p r o t e i n . In c o m p a r a ­ t i v e s t u d i e s a s e c o n d a s s u m p t i o n i s r e q u i r e d - - t h a t the t i s s u e s o r o r g a n s of the d i f f e r e n t s p e c i e s w i l l y i e l d the s a m e p r o p o r t i o n s of a c t i v e p r o t e i n i f the m e t h o d s u s e d a r e the s a m e . T h e r e a r e good r e a s o n s to doubt t h i s , h o w e v e r , c o n s i d e r i n g the p o s s i b i l i t i e s f o r variation in tissue constituents, especially non-specific protein and i n p h y s i c a l t e x t u r e (thus a l t e r i n g g r i n d i n g c o n d i t i o n s ) . T h e s e 1

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC

308

METABOLISM

Ν-demethylation of amwopyrinc (/Amok* formaldehyde produced /$ liver, h) 12

τ

I—ι—r—ι—r—ι—ι—ι—ι—ι—ι—ι—ι—ι July Jan. 196Ô 1969

ι

ι ι July

ι—ι—ι

ι

ι—ι—ι—ι—\—• Jan. 1970

t • July

•

J. H. DeWaide (thesis) Figure 10. Seasonal variation in hepatic drug-metabolizing capacity in the wild roach. Measurements were performed with 9000 g supernatants derived from liver homogenates. Curves are drawn through the median values of the samples (19).

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

TERRiERE

Table VI.

Comparative

Xenobiotic

Metabolism

309

H e p a t i c d r a g o x i d a t i o n i n the r u d d at d i f f e r e n t t i m e s of the y e a r D a t e of a s s a y M a r c h 12, 1970 June 29, 1970 (n = 25) (n = 11)

N - d e m e t h y l a t i o n of amino pyrine activity per— g fresh liver mg l i v e r protein mg l i v e r D N A 100 g b o d y - w e i g h t

/

1.37 0.0119 0.63 2.40

+ + + +

0.52~ 0.0042 0.28 0.86

3.60 0.0379 1.87 10.8

+1.83 + 0.0197 + 0.89 + 6.3

0.41 0.0035 0. 19 0.72

+ + + +

0. 17 0.0015 0.09 0.28

0.87 0.0093 0.47 2.76

+ 0.45 + 0.0046 + 0.25 + 1.67

ρ - H y d r o x y l a t i o n of aniline activity per— g fresh liver mg liver protein mg l i v e r D N A 100 g b o d y - w e i g h t

μΐηοΐββ f o r m a l d e h y d e p r o d u c e d p e r h o u r . b M e a n s w i t h s t a n d a r d d e v i a t i o n . T h e c o r r e s p o n d i n g v a l u e s of the two dates a r e s i g n i f i c a n t l y d i f f e r e n t a t Ρ < 0 . 0 1 ( W i l c o x o n twosided two-sample test). c μΐϊΐοΐββ jD-aminophenol p r o d u c e d p e r h o u r . D a t a f r o m D e W a i d e (19)·

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

XENOBIOTIC METABOLISM

310

f a c t o r s c o u l d l e a d to d i f f e r e n c e s i n the a m o u n t s of s p e c i f i c and n o n - s p e c i f i c p r o t e i n i n the f r a c t i o n f i n a l l y a s s a y e d . I n a s m u c h as the m e t h o d s u s e d f o r p r o t e i n d e t e r m i n a t i o n do not d i s t i n g u i s h b e t w e e n a c t i v e and i n a c t i v e p r o t e i n , the c h a n c e s f o r e r r o r a r e obvious. T h e p o s s i b l e extent of the e r r o r c a n be s e e n i n data f r o m P o h l et a l . (27) who found a 6 - f o l d v a r i a t i o n i n the y i e l d of m i c r o s o m a l p r o t e i n p e r g r a m of l i v e r i n 12 v e r t e b r a t e s p e c i e s . A v a r i a t i o n i n m i c r o s o m a l p r o t e i n content i s r e p o r t e d i n a study of s i x s p e c i e s of w i l d b i r d s (7.0 + 1.3 to 12. 6 _+ 2. 5 mg p r o t e i n per g l i v e r ) (48) and f o r the r a t , r a b b i t , m o u s e , h a m s t e r , and g u i n e a pig ( 2 4 . 0 + 3 . 9 to 35.9 + 10.3 mg p r o t e i n per g l i v e r ) (16). D e W a i d e (19) c o m p a r e and g l u c u r o n i d a t i o n a c t i v i t b a s i s of l i v e r weight, l i v e r p r o t e i n , l i v e r DNA, and body w e i g h t . A s m i g h t be e x p e c t e d , the d r u g m e t a b o l i z i n g a c t i v i t i e s of the l i v e r of the v a r i o u s s p e c i e s r a n k e d d i f f e r e n t l y w h e n e x p r e s s e d i n d i f f e r e n t p a r a m e t e r s . In the N - d e m e t h y l a t i o n of a m i n o p y r i n e , the r a t (average of 30 a n i m a l s ) was 14 t i m e s m o r e a c t i v e than the t r o u t ( a v e r a g e of 30 a n i m a l s ) w h e n c o m p a r e d on the b a s i s of l i v e r weight, 10 t i m e s m o r e a c t i v e on the b a s i s of l i v e r p r o t e i n , 12 t i m e s m o r e a c t i v e on the b a s i s of l i v e r DNA, and 35 t i m e s m o r e a c t i v e on the b a s i s of b o d y w e i g h t . In the h y d r o x y l a t i o n of a n i l i n e , the m u l t i p l e s w e r e 4, 3, 3, and 9 f o r c o m p a r i s o n s on the b a s i s of l i v e r weight, l i v e r p r o t e i n , l i v e r DNA, and b o d y weight. T h i s p r o b l e m has b e e n d i s c u s s e d b y H a r p e r et a l . (50). T h e y p r e f e r to c o m p a r e a c t i v i t i e s b e t w e e n s p e c i e s on the b a s i s of V r a t h e r than s p e c i f i c a c t i v i t i e s and they a l s o show c o m p a r i s o n s on the b a s i s of c y t o c h r o m e P-450 content ( c a t a l y t i c constant, k ^ - ) . T h e b e n z e n e m e t a b o l i s m of l i v e r and lung e n z y m e s f r o m r a t , r a b b i t , and h a m s t e r i s r a n k e d a c c o r d i n g to t h e s e p a r a m e t e r s i n T a b l e VII, c o m p i l e d f r o m t h e i r d a t a . T h e r e i s no a g r e e m e n t b e t w e e n r e l a t i v e a c t i v i t i e s b a s e d o n c y t o c h r o m e P-450 content and those b a s e d on p r o t e i n content o r V . The r e a s o n f o r t h i s i s now u n d e r s t o o d as m e n t i o n e d e a r l i e r i n t h i s symposium. m

a

x

ca

m

a

x

Discussion T h r e e g e n e r a l c o n c l u s i o n s e m e r g e f r o m the f o r e g o i n g r e v i e w of the use of i n v i t r o m e t h o d o l o g y i n s p e c i e s c o m p a r i s o n s : 1) m o s t of the p r o b l e m s o c c u r i n s t u d i e s w i t h a q u a n t i t a t i v e r a t h e r than a q u a l i t a t i v e a s p e c t ; 2) the p r o b l e m s i n c r e a s e as the

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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T a b l e V I I . M i c r o s o m a l B e n z e n e H y d r o x y l a s e A c t i v i t y of R a t , R a b b i t , and H a m s t e r C o m p a r e d i n T h r e e W a y s

Microsomes from

V

5/

B a s i s of C o m p a r i s o n Specific Activity-^

cat

r a t lung

1. 13 + 0 . 2 9

0.49 + 0 . 1 8

-

h a m s t e r lung

3.16 + 0.35

2.41 + 0 . 3 5

27

10.36 + 1.14

4.65+0.44

18

rat l i v e r

1.35 + 0 . 5 5

1.09 ± 0 . 2 7

1.8

hamster liver

9.29+4.03

4.26+0.84

2.2

rabbit liver

3.86 + 0.20

2.08 + 0 . 2 9

1.8

r a b b i t lung

— nmol/min/mg protein ^ n m o l / m i n / m g protein c/ — n m o l /nmol cytochrome P-450/min D a t a f r o m H a r p e r et a l . (50)

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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s y s t e m s b e c o m e m o r e h i g h l y defined; a n d 3) the p r o b l e m s a r e m o r e l i k e l y to o c c u r i n s t u d i e s of i n v e r t e b r a t e s and w i l d v e r t e b r a t e s . It i s a l s o a p p a r e n t that, i n s p i t e of the d i f f i c u l t i e s w h i c h have b e e n i d e n t i f i e d , the m e t h o d has p r o d u c e d m u c h u s e f u l k n o w l e d g e not a v a i l a b l e by o t h e r m e a n s , about s p e c i e s d i f f e r ences. M u c h of the w o r k w h i c h has b e e n done s o f a r i n t h i s a r e a , e s p e c i a l l y that i n v o l v i n g n o n - l a b o r a t o r y a n i m a l s , w o u l d have b e e n i m p r o v e d by m o r e a t t e n t i o n to the u s e of o p t i m u m c o n d i t i o n s . A g e n e r a l r e c o m m e n d a t i o n f o r the f u t u r e u s e of i n v i t r o m e t h o d s i n c o m p a r a t i v e s t u d i e s i s that none of the e x p e r i m e n t a l c o n d i t i o n s o r b i o l o g i c a l f a c t o r s be t a k e n f o r g r a n t e d . T h i s i n c l u d e s the pH a n d t e m p e r a t u r sub-cell fractions, substrat p r e p a r a t i o n , use, s t o r a g e , a n d e n z y m e s o u r c e , the p o s s i b i l i t y of endogenous i n h i b i t o r s , a n d the e f f e c t of a g e a n d s e x . S o m e s u g g e s t i o n s f o r d e a l i n g w i t h s o m e of these p r o b l e m s a r e as follows: 1. C h o i c e of i n v i t r o s y s t e m : T h e c h o i c e of i n v i t r o s y s t e m to b e u s e d i n the c o m p a r i s o n s w i l l depend on the o b j e c t i v e s of the e x p e r i m e n t . W h e n m e t a b o l i c a c t i v i t i e s a r e to be c o m p a r e d q u a n t i t a t i v e l y , the m o r e c o m p l e t e s y s t e m s s u c h a s t i s s u e e x p i a n t s and s l i c e s s h o u l d be u s e d . W i t h these t h e r e i s l e s s danger of l o s i n g i m p o r t a n t e n z y m e s a n d c o - f a c t o r s a n d the c e l l u l a r o r g a n i z a t i o n of the c o n s t i t u e n t s i s p r e s e r v e d . A new a p p r o a c h to s u c h s y s t e m s i s the i n t a c t o r w h o l e c e l l , u s u a l l y p r e p a r e d f r o m the l i v e r a n d thus known a s the h e p a t o c y t e (51). T h i s t e c h n i q u e a p p e a r s to o f f e r s e v e r a l advantages o v e r the l i v e r s l i c e o r e x p i a n t method, m a i n t a i n i n g the o r g a n i z a t i o n a l i n t e g r i t y of the o r i g i n a l t i s s u e w i t h o u t the a r t i f a c t s i n t r o d u c e d b y m e c h a n i c a l i n j u r y to the c e l l s and b y a b n o r m a l i t i e s i n the d i f f u s i o n of s u b s t r a t e o r oxygen. Some r e c e n t u s e s of this t e c h n i q u e a r e d e s c r i b e d below. It i s not p o s s i b l e to p r e p a r e s u c h f r a c t i o n s f r o m s m a l l a n i m a l s , e i t h e r b e c a u s e m e t h o d s a r e not known o r b e c a u s e of the s i z e of the t i s s u e o r o r g a n . In t h e s e c a s e s t h e r e s e e m s no c h o i c e but to u s e a h o m o g e n a t e of the e n t i r e a n i m a l o r a m a j o r body s e g m e n t f o l l o w e d by m i l d c e n t r i f u g a t i o n . A l t h o u g h p a r t i a l l y d i s o r g a n i z e d , this s y s t e m s h o u l d c o n t a i n a l l of the n a t u r a l c o n s t i t u e n t s r e q u i r e d f o r the r e a c t i o n s under study. H o w e v e r , the i n v e s t i g a t o r s s h o u l d c h e c k the r e q u i r e m e n t f o r s u p p l e m e n t a l c o - f a c t o r s s u c h as N A D P H . M o r e h i g h l y d e f i n e d f r a c t i o n s s u c h as m i c r o s o m e s o r m i c r o s o m a l s u p e r n a t a n t a n d s u b f r a c t i o n s of these a r e a p p r o p r i a t e

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w h e n the o b j e c t i v e of the e x p e r i m e n t s i s m o r e q u a l i t a t i v e i n n a t u r e , s u c h as the c o m p a r i s o n of e n z y m e s p e c i f i c i t i e s , s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s , e f f e c t of i n h i b i t o r s a n d a c t i v a t o r s , k i n e t i c s t u d i e s , etc* In t h e s e c a s e s the i n v e s t i g a t o r i s w i l l i n g to s a c r i f i c e o r g a n i z a t i o n and c o m p l e t e n e s s f o r b e t t e r c o n t r o l of e x p e r i m e n t a l c o n d i t i o n s . 2. T e m p e r a t u r e of i n c u b a t i o n ; T h e t e m p e r a t u r e f o r o p t i m u m m e t a b o l i c a c t i v i t y s h o u l d be d e t e r m i n e d f o r e a c h of the s p e c i e s b e i n g c o m p a r e d . W h e n t h i s i s not f e a s i b l e , i t i s proba b l y b e s t to i n c u b a t e e n z y m e s f r o m p o i k i l o t h e r m i c s p e c i e s at o r i n the t e m p e r a t u r e r a n g e of t h e i r n a t u r a l h a b i t a t . 3. E n z y m e k i n e t i c s : W h e n the c o m p a r i s o n s a r e b e i n g m a d e with specific sub-cellula soluble fraction, it is importan s u b s t r a t e b e i n g s t u d i e d i n o r d e r that s a t u r a t i n g c o n c e n t r a t i o n s c a n be u s e d i n e a c h i n c u b a t i o n . W h e n t i s s u e s l i c e s , t i s s u e e x p i a n t s , h e p a t o c y t e s , e t c . , a r e used, K determinations w i l l have l i t t l e v a l u e , s i n c e , p r e s u m a b l y , the s u b s t r a t e conc e n t r a t i o n w i t h i n the c e l l w i l l be d e t e r m i n e d b y p e r m e a b i l i t y a n d d i f f u s i o n p r o p e r t i e s c h a r a c t e r i s t i c of the t i s s u e s b e i n g s t u d i e d . In t h e s e c a s e s , i t i s i m p o r t a n t to m a i n t a i n the s u b s t r a t e l e v e l of the m e d i u m at s a t u r a t i n g c o n d i t i o n s . 4. A g e , sex, a n d s o u r c e of a n i m a l s ; W h e n e v e r p o s s i b l e i n v i t r o c o m p a r i s o n s of m e t a b o l i c a c t i v i t y s h o u l d b e m a d e o n l y a f t e r the r e l a t i o n s h i p b e t w e e n sex, age, a n d d e v e l o p m e n t a l stage and e n z y m e a c t i v i t y have b e e n d e t e r m i n e d . T h e e x p e r i m e n t s s h o u l d then be d e s i g n e d to a c c o m m o d a t e t h e s e v a r i a b l e s . I n the c a s e of s p e c i e s o b t a i n e d f r o m the f i e l d , a t t e n t i o n s h o u l d be g i v e n to e n v i r o n m e n t a l b a c k g r o u n d , e s p e c i a l l y to s u c h f a c t o r s as t e m p e r a t u r e r a n g e of the h a b i t a t and the p o s s i b i l i t y of i n d u c t i o n of e n z y m e s by e n v i r o n m e n t a l p o l l u t a n t s . 5. E n d o g e n o u s i n h i b i t o r s : T h e p r e s e n c e of i n h i b i t o r s w h i c h prevent o r reduce metabolic activity during i n v i t r o assays s h o u l d be a s s u m e d i n l i e u of e v i d e n c e t o the c o n t r a r y w h e n e v e r new s p e c i e s o r new o r g a n s a n d t i s s u e s a r e b e i n g e x a m i n e d . T h i s i s p a r t i c u l a r l y i m p o r t a n t i n s t u d i e s of s m a l l i n v e r t e b r a t e s w h e r e i t i s n e c e s s a r y to h o m o g e n i z e a l l o r p a r t of the a n i m a l i n o r d e r to p e r f o r m the a s s a y . O n e m e t h o d of d e t e c t i n g i n h i b i t i o n i s to i n c u b a t e the s u s p e c t e d f r a c t i o n w i t h one of k n o w n a c t i v i t y f o r e v i d e n c e of i n h i b i t i o n of the l a t t e r . 6. B a s i s of c o m p a r i s o n ; T h e c o m m o n p r a c t i c e of b a s i n g m e t a b o l i c a c t i v i t y o n the p r o t e i n content of the f r a c t i o n b e i n g a s s a y e d i s p r o b a b l y the b e s t w h i c h c a n be d e v i s e d at p r e s e n t . H o w e v e r , s u f f i c i e n t d a t a s h o u l d b e p r o v i d e d to enable o t h e r s to m

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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m a k e t h e i r own e v a l u a t i o n of the a c t i v i t y of the t i s s u e s and o r g a n s c h o s e n f o r study. T h i s i n c l u d e s the y i e l d of p r o t e i n per u n i t w e i g h t of t i s s u e , the t o t a l w e i g h t of o r g a n , t i s s u e , o r a n i m a l , and the p r o t e i n m e t h o d u s e d . W h e n the s p e c i e s b e i n g c o m p a r e d a r e f r o m d i f f e r e n t t a x o n o m i c g r o u p s a t l e a s t one a d d i t i o n a l m e t h o d of e x p r e s s i n g a c t i v i t i e s s h o u l d be p r o v i d e d f o r better c r o s s - r e f e r e n c i n g between l a b o r a t o r i e s . W i t h v e r t e b r a t e s and l a r g e i n v e r t e b r a t e s , a c t i v i t i e s s h o u l d be r e l a t e d to the f r e s h w e i g h t of the t i s s u e s b e i n g s t u d i e d and f o r s m a l l i n v e r t e b r a t e s , to the body w e i g h t . It i s r e g r e t t a b l e that few i n v e s t i g a t o r s of the c o m p a r a t i v e b i o c h e m i s t r y of x e n o b i o t i c s use the s a m e s u b s t r a t e s i n t h e i r s t u d i e s . Often, of c o u r s e w o u l d not p e r m i t t h i s , bu " s t a n d a r d x e n o b i o t i c s c o u l d be u s e d . E x a m p l e s i n c l u d e e f f e c t of i n d u c e r s and i n h i b i t o r s , s e x and age dependency of e n z y m e a c t i v i t y , r e l a t i v e e n z y m e a c t i v i t y , and the o p t i m i z a t i o n of e x p e r i m e n t a l c o n d i t i o n s . T h e u s e of s u c h s t a n d a r d s u b s t r a t e s w o u l d g r e a t l y i m p r o v e c o m m u n i c a t i o n b e t w e e n l a b o r a t o r i e s and thus c o n t r i b u t e to the v a l u e of the i n v i t r o a p p r o a c h . 11

U s e f u l n e s s of the i n v i t r o m e t h o d i n s p e c i e s c o m p a r i s o n s . A l l of the m e r i t s of i n v i t r o m e t h o d s w h i c h have b e e n d i s c u s s e d i n the p r e v i o u s c h a p t e r s a p p l y as w e l l to t h e i r u s e i n species comparisons. These include improved control over e x p e r i m e n t a l c o n d i t i o n s , e l i m i n a t i o n of v a r i a b l e s , i s o l a t i o n and study of s p e c i f i c s y s t e m s , g r e a t e r p r e c i s i o n i n m e a s u r e m e n t s , m o r e f l e x i b i l i t y i n d e s i g n of e x p e r i m e n t s , and e c o n o m i e s of t i m e and l a b o r . I n a d d i t i o n , and i n s p i t e of the g r e a t e r s u s c e p t i b i l i t y to e x p e r i m e n t a l e r r o r m e n t i o n e d e a r l i e r i n t h i s c h a p t e r , the m e t h o d has s o m e s p e c i a l advantages i n s p e c i e s c o m p a r i s o n s . Some examples follow. 1. Studies of m e t a b o l i s m i n w i l d s p e c i e s . I n v i t r o m e t h o d s have a l r e a d y b e e n of v a l u e i n d r a w i n g a t t e n t i o n t o the l o w e r c a p a c i t y of f i s h , r e p t i l e s , a m p h i b i a n s , and o t h e r w i l d s p e c i e s f o r m e t a b o l i z i n g f o r e i g n c o m p o u n d s (3, 6_, 19j 27, 30, 33, 43). It i s not l i k e l y that this i n f o r m a t i o n c o u l d h a v e b e e n g a t h e r e d s o q u i c k l y and o n s u c h a l a r g e s c a l e b y any other m e a n s . T h e d i f f i c u l t i e s of c o l l e c t i n g o r r e a r i n g w i l d a n i m a l s p e c i e s i n s u f f i c i e n t n u m b e r s f o r i n v i v o s t u d i e s and of t r e a t i n g s u c h species w i t h c h e m i c a l s without introducing various s t r e s s e s , a l m o s t e l i m i n a t e s the u s e of s u c h m e t h o d s of e x p e r i m e n t a t i o n . In a d d i t i o n , w i t h a q u a t i c s p e c i e s , t h e r e m a y b e p r o b l e m s w i t h

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Metabolism

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collecting excreted metabolites for identification. Furthermore, these m e t h o d s p e r m i t m o r e u s e of the s a m e a n i m a l s s i n c e s e v e r a l m e t a b o l i c s y s t e m s c o u l d be s t u d i e d w i t h the s a m e o r different organs. 2. M e t a b o l i s m of x e n o b i o t i c s b y h u m a n s ; T h e i n v i t r o m e t h o d has obvious advantages i n s t u d i e s of the m e t a b o l i s m of f o r e i g n c h e m i c a l s b y h u m a n s . W i t h o u t the d i r e c t e x p o s u r e of h u m a n s u b j e c t s , t h e s e m e t h o d s c o u l d p r o v i d e u r g e n t l y needed i n f o r m a t i o n about m e t a b o l i c pathways, i d e n t i t y of m e t a b o l i t e s , e f f e c t of i n h i b i t o r s , d e t e c t i o n of i n t e r a c t i o n s b e t w e e n c h e m i c a l s , l o c a t i o n of s i t e s of m e t a b o l i s m , and the d e t e c t i o n of age and s e x r e l a t i o n s h i p s . S u c h i n f o r m a t i o n c o u l d be o b t a i n e d o n l y w i t h d i f f i c u l t y by other m e a n s of l a b o r a t o r y a n i m a l s , e x p e r i m e n t s into d e c i s i o n s about h u m a n s a f e t y c o u l d b e done w i t h g r e a t e r p r e c i s i o n than at p r e s e n t . 3. U s e i n s c r e e n i n g p r o g r a m s : T h e a u t h o r i s u n a w a r e of the extent to w h i c h i n v i t r o m e t h o d s a r e b e i n g u s e d at p r e s e n t b y the d r u g and p e s t i c i d e i n d u s t r i e s . T h e t e c h n i q u e s s h o u l d be h e l p f u l i n b r i d g i n g gaps b e t w e e n s y n t h e s i s of new c o m p o u n d s a n d their testing for efficacy w i t h l a b o r a t o r y animals, insects, o r p l a n t s . It s h o u l d a l s o be u s e f u l i n g a t h e r i n g i n f o r m a t i o n o n e n v i r o n m e n t a l s a f e t y . In v i t r o s t u d i e s of m e t a b o l i s m b y the e x p e r i m e n t a l o r g a n i s m s m i g h t be h e l p f u l i n d e t e r m i n i n g the r e a s o n s f o r f a i l u r e s i n t o x i c i t y , i n d i c a t i n g w h e t h e r t h i s i s due to inadequate uptake, l a c k of t r a n s p o r t , o r too r a p i d m e t a b o l i s m . The m e t h o d i s a l s o u s e f u l i n d e t e c t i n g u n e x p e c t e d m e t a b o l i t e s or i n p r o d u c i n g m e t a b o l i t e s f r e e of i n t e r f e r i n g c o m p o u n d s . R e s e a r c h needs. The g r e a t e s t o b s t a c l e to the expanded use of i n v i t r o m e t h o d s is the l a c k of e v i d e n c e of t h e i r r e l i a b i l i t y i n e x p l a i n i n g and p r e d i c t i n g i n v i v o e v e n t s . T h i s o b s t a c l e c a n be r e m o v e d o n l y b y a d d i t i o n a l s t u d i e s i n w h i c h i n v i v o and i n v i t r o m e t h o d s a r e c o m p a r e d a s i n the e x p e r i m e n t s r e p o r t e d b y C h i p m a n et a l . (10) and by S u l l i v a n et a l . (11, 12). T h e g r e a t e r use of c o m m o n subs t r a t e s and t o x i c a n t s ( i . e . s t a n d a r d c h e m i c a l s ) b y d i f f e r e n t laboratories would help i n achieving this goal. M o r e w o r k i s needed o n e n z y m e s y s t e m s other than the m i c r o s o m a l o x i d a s e s w h i c h a r e i m p o r t a n t i n the m e t a b o l i s m of x e n o b i o t i c s . T h i s i n c l u d e s the c a r b o x y e s t e r a s e s w h i c h , along w i t h the m i c r o s o m a l o x i d a s e s , a c c o u n t f o r m o s t of the p r i m a r y m e t a b o l i s m of d r u g s and p e s t i c i d e s . M o r e i n f o r m a t i o n i s a l s o

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XENOBIOTIC METABOLISM

needed on the glutathione-S transferases, epoxide hydras e, and other enzymes which are important in the secondary (Type 2) metabolism of xenobiotics. With knowledge of the distribution and properties of these enzymes in various species, the metabolism of xenobiotics can be conducted on a broader scale and thus improve the predictive value of the method. Except for the common laboratory animals, there is a serious deficiency in our knowledge about the rate of development of xenobiotic metabolizing enzymes. It is quite likely that age and stage of development are important factors in these metabolic processes and, until the facts are known, it will be difficult to plan good experiments. Immediate attentio should b give t th f th intact cell technique in comparativ results using rat liver cells have begun to appear and a recent report describes a system using both liver and kidney cells to reconstruct the entire metabolism of a drug from its oxidation and conjugation with GSH to the production of the corresponding mercapturic acid (52). These cells have been shown to resemble the microsomal oxidase system in enzyme activity and substrate specificity. They also appear to contain the conjugation systems of the liver (53-58), and to demonstrate the effect of inducers (52, 57, 59)* Hepatocytes have also been prepared successfully from pig and human liver (60). Methods for the preparation of hepatocytes are described by Mouldes et al. (51). Abstract In addition to the usual doubts about the use of in vitro methods to replace or support those conducted in vivo, their use in comparative biochemistry encounters other uncertainties. These arise from the genetic, behavioral, morphological, and physiological differences among species. There is a need for evidence that, in spite of these special difficulties, in vitro methods can be reliable in detecting metabolic differences between species. The best method of establishing the reliability of data obtained in vitro is to compare the results with those from in vivo experiments performed on the same species. Another test of reliability is to determine how different laboratories rank the same species in terms of their relative enzyme activity. Up to the present time there are only a few reports which permit the use of either of these methods of evaluation. A critical examination of such data indicates that reliable comparisons of xenobiotic metabolism can be made in vitro providing a number of precautions are taken. In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9. TERRiERE

Comparative Xenobiotic Metabolism

317

In vitro studies of species differences are of two types, those in search of quantitative relationships and those seeking qualitative information. There will be fewer problems in studies of the first type if the investigator uses the more complete in vitro systems such as the tissue explant or slice or, in the case of small animals, the low speed centrifugal fraction of the tissue homogenate. When the experiments are qualitative in nature, the more highly defined but less complete sub-cellular fractions are recommended. Special problems arise in the study of wild species. These include: lack of information regarding the relationship between enzyme activity and ag bolic activity; species difference enzyme activity; the presence of endogenous inhibitors of metabolic enzymes; and unpredictable environmental effects on metabolic activity. Another problem encountered in all comparative work but most acute in studies of wild species is the lack of a suitable basis of reference for comparing metabolic activity. The practice of comparing activities on the basis of protein content appears to be the best that can be devised at present, but its limitations should be understood. It is recommended that an additional system of reference be used in most species comparisons. Carefully planned in vitro experiments can be very useful in comparative studies, especially those involving wildlife species which are difficult to rear or manage. The methods should also be useful in screening programs for new drugs and pesticides and in studies of drug metabolism by humans. Research needed to expand the use of these methods includes studies of the use of hepatocytes and other intact cells as substitutes for tissue explants and slices and for sub-cell fractions. Additional information on the age dependency of metabolic enzymes and on conditions affecting in vitro assays for other enzymes such as the carboxyesterases, epoxide hydrase, and the conjugating enzyme systems is also needed.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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XENOBIOTIC METABOLISM

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Gillette, J. R. in "Drug Metabolism: from Microbes to Man" (Parke, D. V. and Smith, R. L . , Eds.) pp. 147168, Taylor and Francis, London, 1977. Quinn, G. P., Axelrod, J., Brodie, Β. Β., Biochem. Pharmacol. (1958) 1, 152. Brodie, Β., Maickel, R. P., and Jondorf, W. R., Fed. Proc. (1958) 17, 1163. Brodie, Β. B., and Maickel, R. P., Proc. First Int. Pharmacol. Meeting (1962) 6, 299. Potter, J. L . , and O'Brien, R. D., Science (1964) 144, 55. Adamson, R. Η. Rall, D. P., Proc Creaven, P. J., Parke, D. V., and Williams, R. T., Biochem. J. (1965) 96, 879. Chakraborty, J., and Smith, J. Ν., Biochem. J. (1964) 93, 389. Schonbrod, R. D., Philleo, W. W., and Terriere, L . C., J. Econ. Entomol. (1965) 58, 74. Chipman, J. K., Kurukgy, M. and Walker, C. Η., Biochem. Pharmacol. (in press). Sullivan, L. J., Chin, Β. Η., and Carpenter, C. P., Tox. and Appl. Pharmacol. (1972) 22, 161. Chin, B. H., Eldridge, J. Μ., and Sullivan, L. J., Clinical Tox. (1974) 7, 37. Hutson, D. H., and Hathway, D. Ε., Biochem. Pharma­ col. (1967) 16, 949. Donninger, C., Hutson, D. Η., and Pickering, Β. Α., Biochem. J. (1972) 126, 701. Chhabra, R. S., Pohl, R. J., and Fouts, J. R., Drug Metab. Disp. (1974) 2, 443. Litterst, C. L . , Mimnaugh, E. G., Reagan, R. L . , and Gram, T. E., Drug Metab. Disp. (1975) 3, 259. Whitehouse, L. W., and Ecobichon, D. J., Pest. Bio­ chem. Physiol. (1975) 5, 314. Machin, A. F., Rogers, Η., Cross, A. J., Quick, M. P., Howells, L. C., and Janes, N. F., Pestic. Sci. (1975) 6, 461. DeWaide, J. H. in "Metabolism of Xenobiotics, " doctoral thesis, Univ. Nijmegen, Drukkerij Leijn, 1971. Castro, J. A. and Gillette, J. R., Biochem. Biophys. Res. Com. (1967) 28, 426.

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Terriere, L . C. and Yu, S. J., Pestic. Biochem. Physiol. (1977) 7, l6l. Yu, S. J. and Terriere, L. C., Pestic. Biochem. Physiol. (1975) 5, 418. Yu, S. J. and Terriere, L. C., Insect Biochem. (1978) ( in press ). Benke, G. M. and Wilkinson, C. F., J. Econ. Entomol. (1971) 64, 1032. Benke, G. M. and Wilkinson, C. F., J. Econ. Entomol. (1972) 65, 1221. Fyffe, J. and Dutton, G. J., Biochim. Biophys. Acta (1975) 411, 41. Pohl, R. J., Bend J. R., Drug Metab Disp (1974) Burns, Κ. Α., Marine Biol. (1976) 36, 5. Elmamlouk, T. H. and Gessner, T., Comp. Biochem. Physiol. (1976) 53C, 57. Buhler, D. R. and Rasmusson, Μ. Ε., Arch. Biochem. Biophys. (1968) 103, 582. Fouts, J. R. and Brodie, Β. Β., J. Pharmacol. Exp. Therap. (1957) 119, 197. Terriere, L . C., Schonbrod, R. D., and Yu, S. J., Bull. W.H.O. (1975) 52, 101. Bend, J. R., James, M. O., and Dansette, P. Μ., Ann. Ν. Y. Acad. Sci. (1977) 298, 505. Elmamlouk, T. H. and Gessner, T., Comp. Biochem. Physiol. (1976) 53C, 19. Krieger, R. I. and Wilkinson, C. F., Biochem. J. (1970) 116, 781. Brattsten, L . B. and Wilkinson, C. F., Comp. Biochem. Physiol. (1973) 45B, 59. Gilbert, M. D. and Wilkinson, C. F., Comp. Biochem. Physiol. (1975) 50B, 613. Schonbrod, R. D. and Terriere, L. C., Pestic. Biochem. Physiol. (1972) 1, 409. Terriere, L . C. and Yu, S. J., Pestic. Biochem. Physiol. (1976) 6, 223. Jordan, T. W. and Smith, J. Ν., Int. J. Biochem. (1970) 139. Kamataki, T. and Kitagawa, Η., Biochem. Pharmacol. (1973) 23, 1915. DeWaide, J. H. and Henderson, P. T., Comp. Biochem. Physiol. (1970) 32, 489.

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319

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XENOBIOTIC METABOLISM

Malins, D. C., Ann. Ν. Y. Acad. Sci. (1977) 298, 482. Gruger, E. H., Wekeli, M. M., Numoto, P. T., and Craddock, D. R., Bull. Environ. Contam. Toxicol. (1977) 17, 512. Yarbrough, J. D. and Chambers, J. E., Life Sci. (1977) 21, 1095. Payne, J. F. and Penrose, W. R., Bull. Environ. Contam. Toxicol. (1975) 14, 112. Payne, J. F., Marine Poll. Bull. (1977) 8, 112. Yawetz, Α., Agosin, M . , and Perry, A. S., Pestic. Biochem. Physiol. (1978) 8, 44. DeWaide, J. H., Comp. Gen. Pharmacol. (1970) 1, 375. Harper, C., Drew Metab. Disp. (1973 Moldeus, P., Hogberg, J., and Orrenius, S., Methods in Enzymology (1978) 52, 60. Moldeus, P., Jones, D. P., Ormstad, Κ., and Orrenius, S., Biochem. Biophys. Res. Comm. (1978) (in press). Inaba, T., Umeda, T., and Mahon, W. Α., Life Sci. (1975) 16, 1227. Vadi, Η., Moldeus, P., Capdevila, J., and Orrenius, S., Cancer Res. (1975) 2083. Grundin, R., Moldeus, P., Vadi, Η., and Orrenius, S., in "Cytochromes P-450 and b " (Cooper, D. Υ., Rosenthal, O., Snyder, R., and Witmer, C., Eds.) Plenum Pub. Corp., New York, N.Y., 1974. Erickson, R. R., and Holtzman, J. L . , Biochem. Pharmacol. (1976) 25, 1501. Wiebkin, P., Ery, J. R., Jones, C. Α., Lowing, R., and Bridges, J. W., Xenobiotica (1976) 6, 725. Burke, M. D., Vadi, Η., Jernstrom, Β., and Orrenius, S., J. Biol. Chem. (1977) 252, 6424. Moldeus, P., Grundin, R., von Bahr, C., and Orrenius, S., Biochem. Biophys. Res. Comm. (1973) 55, 937. Belfrage, P., Borjesson, Β., Hagerstrand, I., Nilsson, Α., Olsson, Α., Wiebe, T., and Akesson, Β., Life Sci. (1975) 17, 1219. 5

56. 57. 58. 59. 60.

RECEIVED

December 20, 1978.

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

INDEX

Absorbance, Type I Absorption of C herbicides in excised leaves root and leaf C Acetyl-JV-acetyltransferase Acetyl-coenzyme A 2-Acetylaminofluorene Acetylation α-1-Acetylmethadol N-Acetyltransferase 14

14

Acheta domesticus

156 4,11/ 8t,9i 9-17 7 203 203 20 203-20 142 203,210

256, 259, 260/,

263/, 267, 278, Activity-structure studies Acyl anilide S-Adenosyl-L- [ methyl- H ] methionine S-Adenosylmethionine Aflatoxin Bi Aglycons 3

Agrostemma githago

165 142

Apis mellifera

251,254

Apoplastic transport Apple

5 41*, 99

Arachis hypogaea

7

Arbutin Arene oxides

116 142 208

Aryl acylamidases Aryl hydroxylation Aryl-2-propynyl ethers Atrazine

279/ 276 Avena fatua 80 Avenu sativa 206 Azasteroids 206 Azobenzenes 143 51/ 42*

Alachor , 22 Alanine 212 Albumin, bovine serum 256 Aldrin 43*, 67/, 87,134, 272* Alkyl alcohols 114 Alkyl thiocyanates 217 1- Alkylimidazoles 280/, 281 Allium canadense

Anion, superoxide Antipyrine

25

94-98 18 275 10,17,21,22,105 ..

7 8

238*, 239/, 240 80 Β

Barban Barley Bathochromic shift Bean Benomyl Benzene hydroxylase activity Benzo(a)pyrene Benzoic acid 1,2,3-Benzothiadiazoles Benzoyl-CoA Benzoylprop-ethyl

Ames test 223 Amiben 8 Amines, azasteroids and nonsteroidal 238* Amino acid conjugates 50 Amino acids, conjugation with 208-214 Beta vulgaris 2- Amino-anthracene 223 Bifenox 4-Aminobenzoic acid 203 Bile cannula, reentry 4-Aminobiphenyl 204 Bioassays 2-Aminofluorene 204 Biphenyl 2-Aminonaphthalene 204 hydroxylase, hepatic l-Aminopropan-2-ol 203 Birds Aminopyrine, N-demethylation of 304/ Blattetta germanica Amitrole 10,17 Blowfly larva Analine hydroxylase, hepatic 287* Boston ivy Analines 80 Botran Bromacil Animal subcellular fractions 149-174,181-233 Buffer(s) Anthranilic acid 116 Butamoxane Anti-12-hydroxy- C-endrin 189 Butylate 14

321

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

108 7,8 160 42-43*, 114 29 312* 142,143 143 275, 277 208 101 42*

101 288 66-68 90,143 297* 192 252

267 41* 83 25 ...188,270 142 22

322

XENOBIOTIC

C D A A (N,N-diallylchloroaeetamide) 113 C - S lyase Ill Caffeic acid 116 Calliphora erythrocephah 267 Callue 95 Canavalie ensiformis 40* Carbamates 250 insecticides 80 Carbanion form of substrates 169 Carbaryl 22,42-43*, 134,135,138,192 metabolism of 65/ (1-naphthyl methylcarbamate) . . . . . 62 toxicity of 278 Carbene form of substrates 170 Carbon monoxide 159/, 160 Carboxylesterase 27 Carcinogens 22 Carrot 7,39-43* Cat 192 Catechol O-methyl transferase 206 Catharanthus roseus 68 Cell culture 137-143 primary 244/ as source of subcellular components 245-246 systems 243-245 techniques 35-71 fraction 302 intact 317 methods, isolated plant part and .... 7-29 techniques, in vitro tissue, organ and isolated 1-30 Centrifugation, differential 182-183 Centrifugation, sucrose density gradient 269 Chicken 141 Chloral hydrate 135 Chloramben 101 Chlorbromuron 22,29 Chlorcyclizine 134,135 Chlorfenvinphos, dealkylation of 295/ 3- Chloro-4-hydroxyaniline 113 Chloro-N-methylaniline 90 p-Chloro-N-methylaniline 87,90 4- Chloro-o-toluidine 139 4-Chlorobiphenyl 134 Chlorofenprop-methyl 99 p-Chloromercuribenzoate 275 Chloropropham 136 Chlorotoluron 139 Chlorphenamidine 139 Chlorpropham 19,22, 111 Chromatogram, DEAE-cellulose 294/, 295/ Chromatography, gel filtration 183-184 Cinnamic acid 89

METABOLISM

Cisanilide 9,39-41*, 45,47/, 48*, 111 Citrus sinensis 41* Clover 42-43* Cockroach 259,267 embryos 244/ leg, cuticle formation in 237* leg, 22,25-dideoxyecdysone in .238*, 242* Coconut milk 36 Collagenase/hyaluronidase 141 Condensations 78 Conjugates 139 Conjugating agent 181 Conjugation 77,103-118,238,240 with amino acids 208-214 with glutathione 142,214-223 with sulfate 142,197-203 Convolvulus arvenis 40* 50 Cotton 7,9,21,25,39-41* p-Coumaric acid 116 Cows 212 tri-o-Cresyl phosphate 275 Cricket/ 259,278 Cucumber 99 Cuticle 4 formation in cockroach leg 237* Cyanophenothrin 275 Cyclizine 135 Cyclodienes 250 Cysteine conjugates Ill Cysteine S-transferase activity 104* Cytochrome P-450 87,143,149-151 Class Β type of 165 clusters 170 cyclic function of 154 cyclic reaction of 173/ ferric 157/ hepatic 296* multiple types of 171-172 reductase, N A D P H 158/ reduction of 158-159 Cytosol 182 Cytotoxicity 137

2,4-D 40-41* D D E , nontoxic 250 DDT 43*, 66,251,252 Dandelion 95,96-97* Dansylamide 142 Daucus carota 7, 39*, 40*, 42* Dealkylation 252 of chlorfenvinphos 295/ N-Dealkylation 80,87 N- and O-Dealkylation thioether 252

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

323

INDEX

O-Dealkylation 87 Dearylation 252 Decarboxylations 78 Dechlorination 252 Dehalogenation 94 Dehydrochlorination 251 N-Demethylase 90 Demthylations 78,142 N-Demethylation 21 of aminopyrine 304/ of monuron 18 Desulfuration 87,252 Detoxication 99 mechanisms 286 in plants 23 N,N-Diallyl-2,2-diehloroacetamide .85,111 Diazinon 272,274/ Diazoxon 273,274 2,6-Dichloro-4-nitrophenol 20 2,4-Dichloroacetophenone 220 Dichlorodiphenylacetic acid ( D D A ) 136 1,2-Dichloronitrobenzene 108 2,4-Dichlorophenacyl chloride 220 2,4-Dichlorophenoxyacetic acid 20,36 2,4-Dichlorophenylethanol 220 3,4-Dichloropropionanilide 44 Diclofop-methyl 9-21 22,25-Dideoxyecdysone in cockroach leg 238* hydroxylation of 241/ metabolism of 235 Dieldrin 67/, 134,193 Differential centrifugation 182-183 Digestion, enzymatic 28 Digestive juices inhibitory 307 5,6-Dihydro-5,6-dihydroxycarbaryl .... 139 l,2-Dihydro-l,2-dihydroxynaphthalene 142 Dihydrodiol 193 Dimefox 136 Dimethoate 101,139 N,N-Dimethyl-2,2-diphenylacetamide 44 cis-2,5-Dimethyl- 1-py rrolidinecarboxanilide 45 Ν,Ν-Dimethylaniline 90 Dimethylvinphos 220 Dinoben ( 2,5-dichloro-3-nitrobenzoic acid) 94 Dinoseb 25 Diphenamid 39-41*, 44,45/, 46*, 116 Diphenylacetic acid 212 Diphenylether 105 Dipropetryn 17 Dismutase, superoxide 165 Dissociation of superoxide 166/ Diuron 20 Dogs Drosophih mehnogaster

204,212 254

Drugs metabolism and toxicity of metabolizing capacity oxidation, hepatic Duochromator Dyfonate

249 181 309/ 310* 153/ 22,206

E P T C (S-Ethyl dipropylthiocarbamate ) 108 α-Ecdysone 245 Ecdysteroid 235,237* Electron the second 166-169 transfer components, microsomal .. 159* transport complexes 172 Enzymatic activity Enzymatic digestion Enzyme activity development induction proteolytic source of stability studies, in vitro substrates for hepatic Epoxidase activities microsomal aldrin Epoxidation microsomal aldrin Epoxide hydratase Ester cleavage Esterases Ether-solubles Ethinimate Ethoxycoumarin Ethylenechlorohydrin Ethylenethiourea Ethylmorphine Excised leaves E y e pigments

270-272 28 269,307 299 162 256 299 303 77-121 306* 261*, 262* 300/, 301/ 87,279/ 263/ 281 142,193,252 252 99-102 52/ 142 143 114 18 142 6 307

F Felidae Fenuron Ferret Ferulic acid Field bindweed Fish Flamprop-isopropyl Flavoprotein Fluometuron Fluoride, phenylmethanesulfonyl

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

192 97 212 116 40-41* 192 101 150/, 256 29,139 256

324

XENOBIOTIC

Fluorodifen

8-10,16,17,38, 40-41*, 94,105 metabolism 26, 27*, 44/ Flurenol butyl ester 87 Foetus 211 Fractionation 265-266 subcellular 182 of microsomal oxidase activity . . . 268/ Freezing, effect on oxidase activity .... 270* Fungicides 1 G G S H conjugates, formation of 106* G S H S-transferase activity 104*, 106* Galium verum 42* Gel filtration chromatography 183-18 Gentiobioside 11 Geranium 41 Gibberellin A 92 Glucose conjugation 114—118 Glucosides 114 Glucosyltransferase 114 Glucuronic acid conjugation 187-197 Glucuronidation 182,192 of methylumbelliferone 193* N-Glucuronide 139 Glucuronyl transferase 142,181,188 Glucose ester 101 N-Glucoside 101 Glutamic acid 212 Glutamine 208,212 γ-Glutamyltransferase 214 Glutathione conjugation 18,23,214-223 S-transferases 107*, 110* X-transferase 103-113 S Glutathione 217 Glycine 208,211,212 N-acylase 208 Glycine max 7,39—43*, 50 Gossypium hirsutum 7, 39* Gromphadorhina portentosa 260/, 263/, 267 Guinea pigs 201, 204,295/ J

35

H Hamster Hank's buffer Harmalol Harmol Helianthus annus Hemeprotein Hens Hepatocyte isolated

204,312* 141 199 199 40* 154 192 313 131,141-143

METABOLISM

Herbicides phenylurea by plant tissue culture, metabolism of Hexamethylbenzene Hippuric acid Homogenization Honey bee Hordeum vulgare Hormones of insects Hornworm larvae House flies Hydratropic acid Hydrogen peroxide Hydrolysis Hydrolytic reactions Hydroperoxidase

1,38-62 139 39-41* 278 208,212 265-266 264*, 269 7 240 240 273 212 166,168/ 77,78 94 85

N-Hydroxyacetanilide 202 N-Hydroxy-2-acetylaminofluorene 198 N-Hydroxy-2-acetyl-aminonaphthalene 202 N-Hydroxy-N-arylaeetamides 202* Hydroxyatrazine 18 Hydroxy benzoic acids 116 4-Hydroxybiphenyl 143 4- Hydroxycarbaryl 135,139 5- Hydroxycarbaryl 139 N-Hydroxy-4-chloroacetanilide 202 7-Hydroxychlorpromazine 189 st/n-12-Hydroxy dieldrin 193 20-Hydroxyecdysone 245 Hydroxylase, hepatic 297* Hydroxylation 78, 87,142, 238, 240, 252 aryl 18 of D D T 252 of 22,25-dideoxyecdysone 241/ N-Hydroxyphenacetin 193,202 Hypsochromic effect 156 Hypsochromic spectral shift 160

I Imidazoles Imipramine Incubation, temperature of Indoleactic acid Induction Inhibition Inhibitors endogenous natural of enzyme activity Inhibitory factors intracellular

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

275 134,135 303 58 99 99 254 287 307 256 262

325

INDEX

Insect age or life stage of the 262 preparation, whole 254, 259 subcellular components 249-281 Insecticides 1,62-70 carbamate 80,274/ by plant tissue culture, metabolism of 42-43* synergists 275-276 In vitro enzyme studies 77-121 methods in species comparisons .287-298 studies, application of 272-277 techniques 285-319 In vivo studies 277-281 Indole 85 Intestines 136 Iodide 84* Ioxynil 8 Iron, ferric 154 Iron—sulfur protein 158 Isonazid 203 Isosuccinimide 0-glucoside 118 Isovanillic acid 116 Isozymes 78,99

Lipophilic drugs 251 Lipophilic zenobiotics 249 Liver 132,136,141 expiants 293 microsomal Cytochrome P-450 .162/, 164/ microsomes, preparation of 152 perfusion apparatus 133/ slices 293,295/ Lubrol 188 Lung 132,136,138 Lycopersicon esculentum

18

Lymphoma cells

138 M

Macremphytus varianus

Malpighia

256

,

Manduca sexta

240

Manometry Matacil Melanization

136 80 254

Meliotus alba

42*

Membrane diagram of the microsomal 170/ fluidity 171 J role of the 170-171 Jackbean 40-41* structure 159 Johnsongrass 105 transport systems 245 Metabolism of 4- C-22,25-dideoxyecdysone 240* Κ of cisanilide 47/ Kelthane 43*, 66 of 22,25-dideoxyecdysone 235 Kidneys 136 of diphenamid 45/ sulphotransferase, bovine 198* fluorodifen 26,27*, 44/ Kinetin 6, 29 of H C E 289/ of herbicides by plant tissue culture 39-41* L in higher plants, pesticide 77-121 in higher plants, xenobiotic 35-71 Lactuca satim 42* of insecticides by plant tissue Leaf culture 42-43* cells, separated 23-28 in leaf discs or sections 21-22 discs 6,19-23 esticide 36 excised y plants, 2,4-D 57/ absorption and translocation in .. 9-17 in rats, hexabarbital 286 radioautographs of soybean 12/ in separated cells 25-28 and roots 7—19 thiocyanage 252 zenobiotic metabolism in 17 of triol 238,240 Lepidopterous larvae 259 in wild species 315 Lettuce 42-43* of xenobiotics 4,7,249-281,285-319 Leucophaea maderae 237 by humans 316 Ligand, soft 160 in insect cell and organ Light retardation of senescence 6 cultures 233-246 Lignification 80 by plant tissue culture 38 Limnephilus 256 Type I 149-174 Lindane 42^43*, 63 Type II 181-223 Lipid peroxidase 307 14

E

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

326

XENOBIOTIC

Metabolites C-carbaryl 64* in cells and medium 46* 2,4-D 51*, 59-61/ 2,4-D-l- C ...54-56* HCE 290* isolation and characterization of .... 23 Metabolizing tissues 234 Metamorphosis, process of 264 Methadone 134,135 8-Methoxybutamoxane 142 N-Methylaniline 90 Methylation 206-207 Methylenedioxyphenyl derivatives .... 275 Methylparathion 217 Methyltransferases 206 4-Methylumbelliferone 143,201 Metobromuron 13 Metribuzin 40-41* Mice 212 Microdissection 28 Microsomal fraction of liver 151 membrane 170 pigments, spectrophotometric analysis of 152-153 Microsomes 188 hepatic 252 Mirex 134 Monkeys 192,204 Monophenyl phosphate 203 Monuron 18-25 Mouse 138,204 Multienzyme complexes 159 Mutagens 222 14

14

Ν N A D P H Cytochrome c reductase 254,256,257/ N A D P H oxidation 254 Naphthalene 142,193 Naphthalene-l,4-diol 139 Naphthalene-l,5-diol 139 Naphthaleneacetic acid 36,116 α-Naphthol 85 1-Naphthol 135,142,192 1- Naphthylacetic acid 212 2- Naphthylacetic acid 212 2-Naphthylamine 203 1-Naphthyl methylcarbamate-iVglucuronide 139 1-Naphthyl ZV-propylcarbamate 275 N I H shift 90 Nicotiana glauca 42* glutinosa 42* sylvestris 42* tabacum 24,40*, 42*

METABOLISM

p-Nitoanisole p-Nitrophenol 4-Nitrophenyl sulphate Nitroreductases Nonsteriodal amines

87,90 134,142,187 199 94 239/

Ο Oat shoots, radioautographs of excised Organ culture systems culture techniques perfusion systems, perfused Organophosphorothioates Organophosphorus compounds

14/ 233 235 132-135 131 89 250

Oxene complex of Cytochrome P-450 169 Oxene type intermediate 169/ Oxenoid. species of oxygen 169 Oxidase activity, effect of freezing on 270* activity, microsomal 259 -like activity 93* mixed function 87-93 Oxidation 77,87,252 inhibition of microsomal 255/ metabolism, H C E 292/ microsomal 260/ NADPH 254 reactions 77-93 Oxycytochrome P-450 163,164/ reduction of 168/ Oxygen, activated 154 Oxygenase, mixed function 142 Oxygenation reactions 166

Ρ P C N B (pentachloronitrobenzene) ... 107 Panax ginseng 69 Papaver somniferum 89 Paraoxon 80,95,134,135 Paraquat 29 Parathion 80,95,134,135 Parenchyma cells 37 Parsley 42-43* Parthenocissus tricuspidata 41*, 62 Pea 8 Peanut 9,26 Pelagonium hortorum 41* Pentachloronitrobenzene 94 Pentamethylbenzene 278 Pentobarbital 134-135 Peptidase 214 Perfluidone 8,18 Perhydroxyl radical 165

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

327

INDEX

Periphneta americana 251, 260/, 267 Peroxidases 62,78-87 Peroxycytochrome P-450 166,168/ Pesticides 249 metabolism 36,77-121,181 Petioles 7 Petroselinum hortense 42-43* Phaseolus radmtus 85 Phaseolus vulgaris .17,43*, 85 Phenols 78, 85,114 Phenothrin 275 3-Phenoxybenzoic acid 212-214 3-Phenoxybenzoyltaurine 214 3-Phenoxybenzoyl chrysanthemum esters 276* Phenylacetic acid 212 Phenylcarbamates 80 Phenylmethanesulfonyl fluoride Phenylurea 80 herbicides 139 Phorate 89 3'-Phosphoadenosine-5'-phosphosulphate 197 Phosphorylation 203 Photophosphorylation, cyclic 6 Photosynthesis 20 inhibition of 106* Phytotoxicity 49,66-68 Picea glauca 62 Pig 192,204 Pisum sativum 8 Placenta 192 Plants discotyledonous 7 enzymes 77,91* monocotyledonous 7 part and cell methods, isolated 7-29 pesticide metabolism in higher 77-121 protoplasts 28-29 radioautographs of oat 15/ radioautographs of soybean 13/ tissue culture 36-38 metabolism of herbicides by 39-41* metabolism of insecticides by .. .42-43* metabolism of xenobiotics by ...... 38 xenobiotic metabolism in higher . 3 5 - 7 1 Phntago major 21 Plantain 21 Plasmalemma 4 Poikilothermic species 314 Polyacrylamide gel electrophoresis 171,172/ Polychlorinated biphenyls 134 Polymorphism 203 Potato 42-43* Precipitation 183 Propachlor 9,108,113 Propanil 22, 39-41*, 44,95,97

Propham Propoxyphene 6-Propyl-2-thiouracil Protein, iron-sulfur Protein synthesis Protoplasts, plant Pseudomonas putida

97,136 142 206 158 6 28-29 151,155/, 160/, 161,163,164/, 165 Purple cockle 42-43* Putidaredoxin 167 Pyrethroid metabolizing esterases 275 Pyridine 206 nucleotide, reduced 150/ Pyroloxygenase 92 Pyrolysis 83

Quinin Quinones

192,254 R

Rabbit 203,204,210-212,312* Radioactivity of 2,4-D 57* Radiochromatograms of 4 - C ecdysteroids 239/ Rat 141,192,201,204,210,212,312* metabolites 294/ Reduction 77 of Cytochrome P-450 158-159 reactions 94-102 Resonance, paramagnetic 155/ Respiration 20 Rice 7,39-41*, 95,96-99* Roach, wild 309/ Roots, excised leaves and 7-19 Ruta graveohns 68 14

Saccharum officianarum Schistocera gregaria Screening programs Selectivity Senescence Sepharose 2B Serine Sex difference Shamouti orange Shrew Simazine Sinapic acid Solarium tuberosum Soret region Sorghum Sorghum vulgare

7 251 316 99 4, 6-7 183 212 286,298 41* 204 8 116 42-43* 162 7,23,105,107* 7

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

328 Soybean 7,16*, 25, 39-43* Spectral shift, hypsochromic 160 Spectrophotometry, difference absorbance 156 Spectroscopy, E P R 156 Spectrum, photochemical action 161/ Spodoptera eridania 256,257/, 260/, 263/, 267 Structure-activity studies 276 Sundangrass 105 Sunflower 40-41* Sugarcane 7,105 Sulfate, conjugation with 197-203 Sulfate and glucuronic acid conjugation 142 Sulfobromophthalein 108 Sulfones 89 Sulfoxide 89,9 Sulfur oxidation 8 Sulphotransferase enzymes 197 Sumithion 95 Superoxide 165/ anion 165 dismutase 165 dissociation of .. 166/ Suspension cultures 49 Symplastic transport 5 Synergistic effect 169 of N A D H 168 Syringic acid 116 Τ Target tissues 234 Taurine 208,211,212 Temperature of incubation 303 Temperature optima 287 Teratogens 222 Tetraethylpyrophosphate 275 Tetraphenylboron 141 Thiocarbamate sulphoxides 218 Thiocyanate metabolism 252 Thione transferases 214 Tissue culture, plant 36-38 culture techniques 35-71 expiants 293,313 maintenance technique 288 metabolizing 234 organ and isolated cell techniques, in vitro 1-30 slices .....136-137 sources 254-264 target 234 Tobacco 36,40-45 hornworm 245 Toruh 262 Toxicity of carbaryl 278

XENOBIOTIC

METABOLISM

Transferases, thione Transferases, G S H Translocation in excised leaves of C herbicides Transpiration stream Transport, apoplastic Transport, symplastic Tree shrew Triazines 5-Triazines 5-Triazinyl mercapturic acid Trichloroacetic acid Trichloroethanol glucuronide Trichloroethylen

214 275 4-7,11/ 9-17 8*, 9* 7 17 5 5 192 9 108 218 135 135 135 134,135

1 4

Triol, metabolic pathways for Triol, metabolism of Triticum aestivum Triticum monococcum Trypan blue exclusion test Trypsin Tulip Tyrosinase system

240 238,240 7 40* 142 141 95,96,97* 256

U Uridine-5'-diphospho-a-D-glucuronic acid

188

V Vanillic acid

116

W Wheat W i l d oat W i l d onion

7,40-41*, 114 7,16* 25

X Xanthommatin 254,255/ Xenobiotic(s) 188 in insect cell and organ cultures, metabolism of 233-246 metabolism of ... 1,4-7,249-281,285-319 in excised leaves 17 in plants 1-30, 35-71 Type I 149-174 Type II 181-223

Ζ Zea mays Zextran Zinia

In Xenobiotic Metabolism: In Vitro Methods; Paulson, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

,

7,40* 80 26